Factor X deletion mutants and analogues thereof

ABSTRACT

Factor XΔ analogues having a deletion of amino acids Arg180 to Arg234 and a modification in the region of the amino acid sequence between Gly173 and Arg179, preparations containing these factor XΔ analogues, and processes for the preparation thereof are described.

[0001] The invention relates to factor XΔ analogues having a deletion ofthe amino acids from Arg180 to Arg234 and a modification in the regionof the amino acid sequence between Gly173 and Arg179, to preparationscontaining the factor XΔ analogues or factor Xa analogues according tothe invention, as well as to methods of preparing the factor XΔanalogues according to the invention.

[0002] After the blood coagulation process has been initiated, thecoagulation cascade continues through sequential activation of variousproenzymes (zymogens) in the blood to their active forms, the serineproteases. Among them are, inter alia, factor XII/XIIa, factor XI/XIa,factor IX/IXa, factor X/Xa, factor VII/VIIa and prothrombin/thrombin. Intheir physiological state, most of these enzymes are only active ifassociated to a membrane surface in a complex. Ca ions are involved inmany of these processes. The blood coagulation will either follow theintrinsic pathway, wherein all protein components are present in theblood, or the extrinsic pathway, wherein the tissue factor plays acritical role. Finally, the wound will close by thrombin cleavingfibrinogen to fibrin.

[0003] The prothrombinase complex is responsible for activatingprothrombin to thrombin. Thrombin is an important enzyme which can actas a procoagulant as well as an anticoagulant. The prothrombinasecomplex, in which, inter alia, factor Va (as cofactor) and factor Xa (asserine protease) are involved, assembles in a Ca-dependent associationat the surface of phospholipids. It is discussed that factor Xa is thecatalytic component of the prothrombinase complex.

[0004] Factor X (Stuart-Prower factor) is a vitamin K-dependentcoagulation glycoprotein which can be activated by the intrinsic and theextrinsic blood coagulation cascade. The primary translation product offactor X (pre-pro-FX) has 488 amino acids and is synthesized by theliver or human hepatoma cells initially as a single chain 75 kDprecursor protein. In plasma, factor X is largely present as a doublechain molecule (Fair et al., 1984, Blood 64:194-204).

[0005] During biosynthesis, after cleavage of the pre-sequence by asignal peptidase (between Ser23/Leu24) and of the propeptide (betweenArg40/Ala41), the single chain factor X molecule is cleaved byprocessing and removal of the tripeptide Arg180-Lys181-Arg182 to thedouble chain form consisting of the approximately 22 kD light chain andthe approximately 50 kD heavy chain, which are connected via a disulfidebridge (FIG. 1). Therefore, factor X circulates in the plasma as adouble chain molecule.

[0006] During the blood coagulation process, factor X is converted frominactive zymogen to active protease factor Xa by limited proteolysis,wherein factor X can be activated to factor Xa in either of twomembrane-associated complexes: in the extrinsic factor VIIa-tissuefactor complex or in the intrinsic factor VIIIa-factorIXa-phospholipid-Ca complex, or “tenase complex” (Mertens et al., 1980,Biochem. J. 185:647-658). A proteolytic cleavage between amino acidsArg234/Ile235 results in the release of an activation peptide having alength of 52 amino acids from the N-terminus of the heavy chain and thusto the formation of the active enzyme, factor Xa. The catalytic centerof factor Xa is located on the heavy chain.

[0007] Activation via the factor VIIa-TF (extrinsic) complex results inthe formation of Factor Xaα (35 kD) and factor Xaβ (31 kD), with apolypeptide of 42 (kD) forming, too, if the factor VIIa concentration inthe complex is low. Factor Xaα is formed by a cleavage at Arg234/Ile235of the heavy chain and represents the activation of factor X to factorXa. The occurence of factor Xaβ presumably results from an autocatalyticcleavage at Arg469/Gly470 in the C-terminus of the heavy chain of factorXaα and the cleavage of a 4.5 kD peptide. Like factor Xaα, factor Xaβhas catalytic activity. It has been shown, however, that a plasminogenreceptor binding site is formed by the cleavage of factor Xaα to factorXaβ, and that factor Xaβ optionally has fibrinolytic activity or isinvolved in fibrinolysis as a cofactor. The transformation of factor Xaαto factor Xaβ, however, is slower than the formation of thrombin, thuspreventing the initiation of fibrinolysis before a blood clot is formed(Pryzdial et al., 1996, J. Biol. Chem. 271:16614-16620; Pryzdial et al.,1996, J. Biol. Chem. 271:16621-16626).

[0008] The 42 kD polypeptide results from processing in the C-terminusof the heavy chain between Arg469/Gly470 without previous processingbetween Arg234/Ile235. Like a factor Xaγ fragment formed by proteolysisat Lys370, this intermediate has no catalytic activity (Mertens et al.,1980, Biochem. J. 185:647-658; Pryzdial et al., 1996, J. Biol. Chem.271:16614-16620).

[0009] Intrinsic factor X activation is catalysed by the factorIXa-factor VIIIa complex. The same processing products are obtainedduring activation, but the factor Xaβ product is obtained in a largerquantity than other factor X processing products (Jesty et al., 1974, J.Biol. Chem. 249:5614).

[0010] In vitro, factor X can, for instance, be activated by Russell'sviper venom (RVV) or trypsin (Bajaj et al., 1973, J. Biol. Chem.248:7729-7741) or by purified physiological activators, such as FVIIa/TFcomplex or factor IXa/factor VIIIa complex (Mertens et al., 1980,Biochem. J. 185:647-658).

[0011] Most commercially available factor X products from plasma containa mixture of factor Xaα and factor Xaβ, because after activation offactor X to factor Xa mainly factor Xaα is formed, which is, in turn,cleaved to factor Xaβ in an autocatalytic process.

[0012] In order to produce a uniform factor Xa product having highmolecular integrity, EP 0 651 054 suggested to activate factor X withRVV over an extended period of time so that the resulting final productsubstantially contains factor Xaβ. The by-products, e.g. factor Xaα, aswell as the protease were subsequently removed by severalchromatographic steps.

[0013] cDNA for factor X has been isolated and characterized (Leytus etal., 1984, Proc. Natl. Acad. Sci., U.S.A., 82:3699-3702; Fung et al.,1985, Proc. Natl. Acad. Sci., U.S.A., 82:3591-3595). Human factor X hasbeen expressed in vitro in various types of cells, such as humanembryonal renal cells or CHO cells (Rudolph et al., 1997., Prot. Expr.Purif. 10:373-378, Wolf et al., 1991, J. Biol. Chem. 266:13726-13730).However, it was found that in the recombinant expression of human factorX, the processing at position Arg40/Ala41 is inefficient, as opposed tothe situation in vivo, and that different N-termini form at the lightchain of factor X (Wolf et al., 1991, J. Biol. Chem. 266:13726-13730).Recombinant factor X (rFX) was activated to rfactor Xa (rFXa) by RVV invitro, or rFXa was expressed directly, with the activation peptide beingdeleted from amino acid 183 to amino acid 234 and replaced by atripeptide in order to allow processing directly to a double chain rFXaform. About 70% of purified rFX was processed to light and heavy chain,while the remaining 30% represented single chain rFX of 75 kD. Directexpression of rFXa did result in the formation of active factor Xa, butalso of inactive intermediates. Furthermore, Wolf et al. (1991, J. Biol.Chem. 266:13726-13730) detected still reduced activity of recombinantfactor X, which they ascribed to the poorer ability of rFX to beactivated by RVV and to the inactive protein and polypeptide populationsof the single chain precursor molecule. In particular, they found highrFXa instability when expressed by recombinant cells, which theyascribed to the high rate of autoproteolysis.

[0014] In order to study the function of the C-terminal peptide offactor Xaα, Eby et al. (1992, Blood 80 (suppl. 1): 1214 A) introduced astop codon at position Gly430 of the factor X sequence. However, theydid not find a difference between the rate of activation of factor Xa(FXaα) with β-peptide or a deletion mutant without β-peptide (FXaβ).

[0015] Factor Xa is an important component of the prothrombinase complexand is therefore under discussion as a primary mediator for quickhemostasis, and thus it seems suitable for the treatment of patientssuffering from blood coagulation disorders, e.g. hemophilia.

[0016] Particularly the treatment of hemophilia patients suffering fromfactor VIII or factor IX deficiency with factor concentrates producedfrom plasma is often complicated by the formation of inhibitingantibodies against these factors in long-term therapy. Therefore, anumber of alternatives have been developed to treat hemophiliacs withfactors having bypass activity. The use of prothrombin complexconcentrate, partially activated prothrombinase complex (APPC), factorVIIa or FEIBA has been suggested. Commercial preparations having factorVIII bypass activity (FEIBA) are, for instance, FEIBA® or Autoplex®.FEIBA, contains comparable units of factor II, factor VII, factor IX,factor X and FEIBA, small amounts of factor VIII and factor V, andtraces of activated coagulation factors, such as thrombin and factor Xaor a factor having factor X-like activity (Elsinger, 1982, ActivatedProthrombin Complex Concentrates. Ed. Mariani, Russo, Mandelli, pp.77-87). Elsinger particularly points at the importance of a “factorXa-like” activity in FEIBA. Factor VIII bypass activity was shown byGiles et al (1988, British J. Haematology 9:491-497) for a combinationof purified factor Xa and phospholipids in an animal model.

[0017] Therefore, factor X/Xa or factor X/Xa-like proteins, either aloneor as a component of a coagulation complex, are in high demand and canbe used in various fields of application in hemostasis therapy.

[0018] In vivo as well as in vitro, the half-life of factor Xa isconsiderably shorter than the half-life of the zymogen. For instance,factor X can be stored stably in glycerol for 18 months, while factor Xais stable for only 5 months under the same conditions (Bajaj et al.,1973,. J. Biol. Chem. 248:7729-7741) and shows reduced activity by morethan 60% after 8 months in glycerol at 4° C. (Teng et al., 1981,Thrombosis Res. 22:213-220). The half-life of factor Xa in serum is amere 30 seconds.

[0019] Because factor X is instable, the administration of factor Xpreparations has been suggested (U.S. Pat. No. 4,501,731). If, however,the bleeding is so serious that the patient might die, particularly in ahemophiliac, the administration of factor X is ineffective, becauseowing to the functional “tenase complex” deficiency in the intrinsicpathway of blood coagulation, factor X can not be sufficiently activatedto factor Xa, and activation via the extrinsic pathway is often too slowto show effects quickly. Moreover, hemophiliacs have sufficient amountsof factor X, but its prothrombinase activity is 1000 times less thanthat of factor Xa. In such cases it is necessary to administer activatedfactor Xa directly, optionally in combination with phospholipids, asdescribed in Giles et al. (1988, British J. Haematology 9:491-497) orwith other coagulation factors, e.g. with factor VIII bypass activity.

[0020] In the preparation of factor Xa from factor X, activation so farmostly has been carried out by non-physiological activators of animalorigin, such as RVV or trypsin, and it was necessary to make absolutelysure that the final product is completely free of these proteases. Asmentioned above, when factor X is activated to factor Xa, quite a numberof inter-mediates, some of them inactive, are formed (Bajaj et al.,1973, J. Bio. Chem. 248:7729-7741; Mertens et al., 1980, Biochem. J.185:647-658). The presence of such intermediates results in reducedspecific activity of the product and may produce intermediates which canfunction as active serine protease antagonists. Therefore, thepreparation of a uniform, pure product having high specific activityaccording to conventional methods requires complex processes ofactivation and chromatographic purification.

[0021] Thus, the aim of the present invention is to provide apreparation containing a polypeptide having factor X/Xa activity whichexhibits high stability and can be activated to factor Xa without usingany of the usual proteases, particularly those of animal origin, suchas, for instance, RVV or trypsin. Another aim is to provide apharmaceutical preparation having factor VIII bypass activity.

[0022] According to the present invention, the aim is reached byproviding a factor X analogue having a deletion of the amino acidsArg180 to Arg234 of the factor X amino acid sequence and a modificationof this factor X deletion mutant in the region of the amino acidsequence between Gly173 and Arg179. By the deletion of the amino acidsequence from Arg180 to Arg234, the tripeptide Arg180 to Arg182 as wellas the activation peptide Ser183 to Arg234 are deleted, and the lightand heavy chains of factor X and the amino acids Arg179 and Ile235 aredirectly fused. This fusion sequence, however, does not contain anatural cleavage site for a protease. By modifying the region of thefactor X sequence between amino acid Gly173 and Arg179 and optionally ofIle235, a factor X deletion mutant according to the present invention isobtained, which has a novel detection and processing site not occurringat this position in the polypeptide for a protease which would notusually cleave the polypeptide at this position. Said modification is,at least, an exchange of at least one amino acid between position Gly173and Arg179 and optionally of Ile235 of the factor X amino acid sequence.The position of amino acids refers to the numbering according to thesequence presented in FIG. 1, starting with Met1 and ending with Lys488.In order to simplify the nomenclature, the amino acid numbering givenfor the complete factor X sequence is adhered to for the modified factorX deletion mutant according to the present invention, but said modifiedfactor X deletion mutant will hereinafter be referred to as factor XΔanalogue.

[0023] Said modification can be a substitution of at least one aminoacid, or an insertion of a peptide sequence representing a proteaserecognition or cleavage site. In the factor XΔ analogue according to thepresent invention, the modification is preferably such that itrepresents a recognition and cleavage sequence for a protease from thegroup of endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2,PC4, PACE 4, LPC/PC7 (as described in Barr et al., 1991,. Cell 66:1-3 orin U.S. Pat. No. 5,460,950), serine proteases, such as factor IIa,factor VIIa, factor IXa, factor XIIa, factor XIa, factor Xa, orkallikrein, or a derivative of these proteases.

[0024] Preferably, said modification is selected such that processing byone of these proteases leads to a polypeptide corresponding to nativefactor Xa in its biological activity and displaying factor Xa activity.For optimal processing, it may be necessary in individual cases toexchange the amino acid Ile235, too. Preferably, however, theNH₂-terminal amino acid isoleucine of the heavy chain should still bemaintained after activation, because isoleucine represents one of thoseamino acids which perform an essential function in the formation of thesubstrate binding pocket (Watzke et al., 1995, Molecular Basis ofThrombosis and Hemostasis, ed. Katherine High & Harold Roberts). Thefactor XΔ analogues according to the present invention display astructural difference, particularly on the amino acid level, as comparedto a native factor X sequence, but after activation their activity iscomparable to that of naturally occurring factor X or factor Xa,respectively.

[0025] The invention exemplary provides a number of factor XΔ analogueshaving a deletion and, in addition, a modification between Gly173 andArg179 and optionally of Ile235. Modifications can be at one or morepositions in the region between amino acids Gly173 and Arg179, andoptionally Ile235, based on the factor X sequence numbered from Met1 toLys488 according to FIG. 1. Amino acid substitutions can be at positionsIle235 (R1), Arg179, Glu178 (R2), Leu177 (R3), Thr176 (R4), Gln175 (R5)and Lys174 (R6), with Arg179, however, preferably remaining unchanged.

[0026] Preferably, the factor XΔ analogues according to the inventioncontain a factor X sequence with Gly173-R6-R5-R4-R3-R2-Arg179-R1,wherein R1=Ile, Val, Ala, Ser or Thr; R2 Glu, Thr, Pro, Gly, Lys or Arg;R3=Leu, Phe, Lys, Met, Gln, Ser, Val, Arg or Pro; R4=Thr, Asn, Asp, Ile,Ser, Pro, Arg or Lys; R5=Asn, Lys, Ser, Glu, Gln, Ala, His or Arg; andR6=Arg, Asp, Phe, Thr, Leu or Ser.

[0027] Preferred embodiments of the factor X analogues according to theinvention are factor X analogues having a modification with

[0028] a) R1=Ile, R2=Thr, R3=Leu, R4=Asn and optionally R5=Asn and/orR6=Asp, and processed by factor VIIa or factor IXa;

[0029] b) R1=Val, R2=Thr, R3=Phe, R4=Asp, and optionally R5=Asn and/orR6=Phe and/or R1=Ile or Val (FIG. 2A), and processed by factor XIa;

[0030] c) R1=Ile or Val, R2=Phe, R3=Lys, R4=Ile, and optionally R5=Lysand/or R6=Thr (FIG. 2C), or

[0031] R1=Ile, R2=Thr, R3=Ser, R4=Thr, and optionally R5=Lys and/orR6=Thr (FIG. 2I), and processed by factor XIIa;

[0032] d) R1=Ile or Val, R2=Thr, R3=Met, R4=Ser, and optionally R5=Serand/or R6=Leu (FIG. 2D), and processed by kallikrein;

[0033] e) R1=Ile, R2=Gly, R3=Gln, R4=Pro, and optionally R5=Lys and/orR6=Ser (FIG. 2H), or

[0034] R1=Ile, R2=Gly, R3=Glu, R4=Ile (FIG. 2F), or

[0035] R1=Ile, R2=Thr, R3=Lys, R4=Met (FIG. 2E), and processed by factorXa;

[0036] f) R1=Ile, R2=Lys, R3=Arg, R4=Arg, and optionally R5=Glu and/orR6=Leu, or

[0037] R1=Ile, R2=Thr, R3=Val, R4=Arg, and optionally R5=Ala and/orR6=Leu, or

[0038] R1=Ile, R2=Arg, R3=Val, R4=Arg, and optionally R5=Gln and/orR6=Leu, or

[0039] R1=Ile, R2=Arg, R3=Arg, R4=Arg, and optionally R5=His and/orR6=Leu, or

[0040] R1=Ile, R2=Lys, R3=Pro, R4=Arg, and optionally R5=Asn and/orR6=Leu, or

[0041] R1=Ile, R2=Lys, R3=Arg, R4=Ile, and optionally R5=Arg and/orR6=Leu, or

[0042] R1=Ile, R2=Lys, R3=Ser, and R4=Arg, or

[0043] R1=Ile, R2=Thr, R3=Val, and R4=Arg, or

[0044] R1=Ile, R2=Lys, R3=Leu, and R4=Arg (all see FIG. 2G),

[0045] with the sequences mentioned under f) being processed by adibasic endoprotease, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4,PACE 4, LPC/PC7, or a derivative of these proteases.

[0046]FIG. 2 shows a possible selection of modifications and amino acidexchangers leading to changed protease specificity.

[0047] The modifications can be carried out by, for instance, directedin vitro mutagenesis or PCR or other methods of genetic engineeringknown from the state of the art which are suitable for specificallychanging a DNA sequence for directed exchanges of amino acids.

[0048] According to the present invention, the factor XΔ analogue of theinvention is preferably activated to a factor Xa analogue by a proteaseselected from the group of endoproteases, such as kexin/Kex2,furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7, serine proteases, suchas factor IIa, factor VIIa, factor IXa, factor XIIa, factor XIa, factorXa, or kallikrein, or a derivative of these proteases.

[0049] The factor XΔ analogues according to the invention are present assingle chain polypeptides in enzymatically inactive form. Active factorXa analogues are only obtained by cleavage by a protease to the doublechain form. Thus, the modification allows activation of the inactive,single chain factor XΔ analogue polypeptide to the double chain activeform.

[0050] One of the difficulties in the preparation of active factor Xa isits instability, because autocatalysis results in the formation ofother, inactive intermediates besides factor Xaα and factor Xaβ.

[0051] For the preparation of essentially intact, active factor X/Xa andfactor X/Xa-like molecules, respectively, it would therefore bedesirable to obtain only such proteins as result in stable finalproducts.

[0052] It is well known that a preferred cleavage site for theprocessing of factor Xaα (FXaα) to factor Xaβ (FXaβ) is betweenArg469/Gly470. Based on research by Eby et al. (1992, Blood. Vol. 80,Suppl. 1, 1214), next to a prominent carboxy-terminal peptide (aminoacid residues 476-487) of factor X, another, shorter peptide (amino acidresidues 474-477) is found which is formed by autocatalysis of factorXaα. In order to focus directed processing of intact factor X toessentially active factor Xa without obtaining inactive processingintermediates, the factor XΔ analogues of the invention optionally havefurther modifications.

[0053] Therefore, according to a particular embodiment, the factor XΔanalogues according to the invention have one further modification inthe C-terminal region of the factor X amino acid sequence.

[0054] According to one embodiment, a factor XΔ analogue as describedabove has an intact β-peptide (FXΔa). The factor XΔ analogues accordingto the invention particularly have a modification in the region of theC-terminal β-peptide cleavage site which prevents cleavage of theβ-peptide from factor X after activation of factor XΔ to factor Xaanalogue. Thus a factor Xa molecule is obtained which can be isolated upto 100% as intact factor Xaα molecule.

[0055] Said modification can be a mutation, deletion or insertion in theregion of the factor X amino acid sequence between amino acid positionArg469 and Ser476 and optionally of Lys370. However, an amino acidsubstitution is preferred which prevents the polypeptide from folding asa consequence of the amino acid exchange, which would influence thestructure and thus possibly the function and activity of the protein.

[0056] According to one embodiment, the factor XΔ analogues of theinvention have one of the amino acids at position Arg469 and/or Gly470exchanged, with Arg469 being preferably exchanged for Lys, His or le,and Gly470 being preferably exchanged for Ser, Ala, Val or Thr.

[0057] Besides a mutation at position Arg469 and/or Gly470, the factorXΔ analogues according to the invention can have a further mutation atposition Lys370 and/or Lys475 and/or Ser476. Amino acid substitution atthis (these) position(s) prevents processing of factor Xaα analogue tofactor Xaβ analogue or C-terminal truncated factor Xa analogues,respectively, because the natural occurring sequence(s) is (are)modified such that an occasional autocatalytic cleavage of acarboxy-terminal peptide becomes impossible.

[0058] According to a different embodiment, the factor X analogues ofthe invention have deleted carboxy terminal β-peptide (FXΔβ). Such afactor X analogue can be prepared by expressing a cDNA coding for factorXΔ analogue in a recombinant expression system, cloning only thosesequences that code for the amino acids Met1 to Arg179/Ile235 to Arg469.

[0059] According to a further embodiment, the factor XΔ analoguesaccording to the invention have a translation stop signal in theC-terminal region of the factor X sequence. This translation stop signalis preferably located at a position following a C-terminal amino acidformed after natural processing. Therefore, the translation stop signalis preferably at the position of amino acid 470 of the factor Xsequence, so that the terminal Arg469 of factor XΔβ is retained. Forthis purpose, the codon GGC encoding the amino acid Gly470 issubstituted by TAA, TAG or TGA.

[0060] Another aspect of the present invention relates to factor XΔanalogues which are activated to factor Xa analogues by treatment withan appropriate protease in vitro, i.e. the activated factor XΔanalogues. Depending on the factor XΔ analogue used and activated, afactor XaΔ analogue is obtained which, at the C-terminal end of thelight chain, has corresponding amino acid modifications, as compared tothe natural factor Xa sequence. According to the invention, thesemodifications are, however, selected in such a way as not to negativelyaffect the biological activity.

[0061] If such a factor X analogue additionally has a translation stopsignal in the C-terminal region of the β-peptide, modified factor Xaβmolecules are obtained. If, however, a factor X analogue is employedwhich has modification(s) within the β-peptide sequence resulting in theβ-peptide not being cleaved off, a factor Xaα analogue with an aminoacid exchange in the C-terminus of the molecule is obtained.

[0062] The factor XΔ analogues according to the invention only havemodifications which change the specificity for the ability to beactivated and do not significantly influence the activity. Therefore, inany case, biologically and functionally active factor Xa molecules orfactor Xa analogues, respectively, are obtained.

[0063] In vitro activation can be effected by a protease selected fromthe group of endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3,PC2, PC4, PACE 4, LPC/PC7, serine proteases, such as factor IIa, factorVIIa, factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, ora derivative of these proteases. It is within the scope of the presentinvention to use any protease, except RVV or trypsin, as long as it isapt to process the factor XΔ analogue according to the invention tofactor Xa analogue.

[0064] Although Wolf et al. (1991, J. Biol. Chem. 266:13726-137309), forinstance, have assumed that an endopeptidase, such as Kex2, furin orPACE, is involved in the processing of the factor Xa deletion mutantdescribed by this group, they do not give a hint as to the influence ofone of these proteases on the processing of factor X. Similarly, U.S.Pat. No. 5,660,950 describes the recombinant preparation of PACE and theuse of the protease to improve processing of vitamin K dependentproteins. In a long list of blood factors, factor X is mentioned amongothers, but no data are provided to verify this statement.

[0065] The present invention demonstrates unambiguously for the firsttime that a protease necessary for the maturing process of factor X is adibasic endoprotease, particularly endogenic furin. In vivo, theendoprotease mainly mediates the cleavage of the single chain factor Xmolecule to the mature form consisting of heavy and ligth chain. Invitro, it also mediates the cleavage of the factor X propeptide sequence(Example 2).

[0066] According to a particular embodiment, a factor XΔ analogue isprovided which is preferably present in purified form as a single chainmolecule. Factor XΔ analogues having in the modified region a cleavagesite for a protease not present in recombinant cells are obtained afterexpression as single chain molecules. The single chain factor XΔmolecule is particularly characterized by high stability and molecularintegrity. So far, a single chain, inactive factor XΔ molecule could notbe isolated in purified form, because in recombinant cells it isprocessed to factor Xa and a number of other, also inactive,intermediates (Wolf et al., 1991, J. Biol. Chem. 266:13726-13730). Theisolated single chain factor XΔ analogue can be activated by specificprocessing directly to the double chain factor Xa analogue form. Thiscan be effected by bringing a single chain factor XΔ molecule isolatedfrom a recombinant cell into contact with a protease cleaving theactivation site present in the factor XΔ analogue. If, for example, afactor XΔ analogue having a furin activation site is expressed in afurin deficient cell, it can be isolated as a single chain factor XΔanalogue and processed to an active, double chain factor XΔa analogue bybringing it into contact with a dibasic protease, such as furin/PACE orKex2. Factor XΔ analogues having a processing site for serine proteaseor kallikrein can also be isolated as single chain molecules in furinexpressing cells and then processed with the serine protease to activefactor Xa analogues.

[0067] Due to the selective and directed processing reaction, a factorXa analogue thus obtained has high stability and structural integrityand, in particular, is free of inactive factor X/Xa analogueintermediates and autoproteolytic decomposition products.

[0068] According to the present invention, the factor XΔ analogue of theinvention is provided in the form of a factor XΔa having intactβ-peptide as well as in the form of a factor XΔ analogue having adeletion of the β-peptide.

[0069] Another aspect of the present invention relates to recombinantDNA encoding the factor XΔ analogues of the invention. Said recombinantDNA results after expression in a factor XΔ analogue with an amino acidsequence corresponding to human factor X except for a deletion of aminoacids from Arg180 to Arg234 and a modification allowing processing andactivation to active factor Xa analogues having both intact as well asdeleted β-peptide.

[0070] A further aspect of the invention relates to a preparationcontaining a purified factor XΔ analogue having a deletion of aminoacids from Arg180 to Arg234 and a modification of amino acids in theregion between Gly173 and Arg179 and optionally of Ile235. Saidmodification leads to a novel recognition or cleavage site not naturallylocated at this position in the polypeptide for a protease which usuallydoes not process the polypeptide at this position. Said preparation canbe a purified preparation containing single chain factor XΔ analogue,the polypeptides being obtained from a cell culture system either afterisolation from the cell culture supernatant or from a cell cultureextract. A recombinant factor XΔ analogue prepurified from a cellculture system can be further purified by methods known from the priorart. Chromatographic methods are particularly useful for this purpose,such as gel filtration, ion exchange or affinity chromatography.

[0071] According to one embodiment, the preparation according to theinvention contains the factor XΔ analogue as a single chain molecule inenzymatically inactive form, with the factor XΔ analogue having a purityof at least 80%, preferably at least 90%, particularly preferably atleast 95%, and the purified preparations containing no inactive,proteolytic intermediates of factor X/Xa analogues.

[0072] According to a particular aspect, the preparation contains singlechain factor XΔ analogue having a modification allowing activation tofactor Xa analogues by one of the proteases selected from the group ofdibasic endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2,PC4, PACE 4, LPC/PC7, serine proteases, such as factor IIa, factor VIIa,factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or aderivative of these proteases. The activation is effected by bringingthe factor XΔ analogue into contact with the appropriate protease, whichcleaves at the modified sequence, whereby a factor Xa analogue isobtained.

[0073] In the preparation according to the invention, the factor XΔanalogue can be present either as factor XΔα (FXΔα) having intactβ-peptide, or as factor XΔβ having a deletion of the β-peptide or otherC-terminal deletions.

[0074] According to a further embodiment, the preparation according tothe present invention contains the factor XΔ analogue preferably as asingle chain molecule in isolated form. For this purpose, factor XΔanalogue is obtained, for instance, by recombinant preparation, as asingle chain molecule having one modification allowing activation tofactor Xa analogue in vitro. The activation of factor XΔ analogue tofactor Xa analogue can be effected by bringing factor X analogue intocontact with a protease selected from the group of dibasicendoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE4, LPC/PC7, serine proteases, such as factor IIa, factor VIIa, factorIXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or a derivativeof these proteases. The protease can be immobilized on a carrier.

[0075] The preparation according to the invention can serve as astarting material for the production and recovery of factor Xaanalogues. For large-scale production, the preparation containing singlechain factor XΔ analogue is brought into contact with an optionallyimmobilized protease under conditions allowing optimal activation offactor XΔ analogue to factor Xa analogue, and factor Xa analogues areobtained. The factor Xa analogue thus recovered can subsequently bepurified by generally known methods and formulated to a pharmaceuticalcomposition having factor Xa activity.

[0076] According to a further aspect of the present invention, apreparation is provided containing a factor Xa analogue having highstability and structural integrity, which is particularly free ofinactive factor X/Xa analogue intermediates and autoproteolyticdecomposition products. It is obtainable by activating a factor XΔanalogue of the above-defined type and preparing a correspondingpreparation.

[0077] According to a particular embodiment, the preparation containingthe purified, single chain or double chain factor XΔ analogue contains aphysiologically acceptable carrier and is optionally formulated as apharmaceutical preparation. The formulation can be effected according toa method common per se, and it can be mixed with a buffer containingsalts, such as NaCl, CaCl₂, and amino acids, such as glycin and/orlysin, at a pH in the range of 6 to 8 and formulated as a pharmaceuticalpreparation. The purified preparation containing factor X analogue canbe provided as a storable product, as a ready-made solution,lyophilisate or deep frozen until final use. Preferably, the preparationis stored in lyophilized form and dissolved with an appropriatereconstitution solution to an optically clear solution.

[0078] However, the preparation according to the present invention canalso be provided as a liquid preparation or in the form of deep frozenliquid.

[0079] The preparation according to the invention is particularlystable, i.e. it can be left standing in dissolved form over an extendedperiod of time before application. It has appeared that the preparationaccording to the invention suffers no loss in activity for several hoursup to days.

[0080] The preparation according to the invention can be provided in anappropriate device, preferably an application device, in combinationwith a protease selected from the group of endoproteases, such askexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7, serineproteases, such as factor IIa, factor VIIa, factor IXa, factor XIIa,factor XIa, factor Xa, or kallikrein, or a derivative of theseproteases.

[0081] The preparation according to the invention containing a factor XΔanalogue in combination with a protease able to activate the factor XΔanalogue to factor Xa analogue can be provided as a combinationpreparation consisting of a vessel containing a protease immobilized ona carrier, optionally in the form of a small column or a syringe chargedwith an immobilized protease, and a vessel containing the pharmaceuticalpreparation with factor XΔ analogue. For activation of the factor XΔanalogue, the solution containing the factor XΔ analogue is pressed overthe immobilized protease, for instance. During storage of thepreparation, the solution containing factor XΔ analogue is preferablykept apart from the immobilized protease. The preparation according tothe invention can be present in the same vessel as the protease, withthe components, however, being separated in space by an impermeableseparation wall which can be easily removed to use the product. Thesolutions can also be stored in individual vessels and brought intocontact only shortly before application.

[0082] In a particular embodiment, the protease used for activation is aserine protease naturally involved in blood coagulation, such as factorXIIa, which need not be separated from the activated factor Xa analoguebefore application but can be applied together with it.

[0083] Factor XΔ analogue can be activated to factor Xa analogue shortlybefore direct use, i.e. before application to the patient. Theactivation can be effected by bringing it into contact with animmobilized protease or by mixing solutions containing a protease on theone hand and factor XΔ analogue on the other. Thus, it is possible tokeep the two components in solution separately and to mix them by meansof an appropriate device wherein the components get into contact witheach other while passing through, and thus to activate factor XΔanalogue to factor Xa analogue. The patient will be administered amixture of factor Xa and another serine protease which has effected theactivation. Particular care has to be taken as regards the dosage,because endogenous factor X is activated by the additionaladministration of a serine protease, which might result in shorterclotting time.

[0084] According to a preferred embodiment, the pharmaceuticalpreparation is provided in an appropriate device, preferably anapplication device, either in frozen liquid or in lyophilized form. Anappropriate application device can be a double compartment syringe asdescribed in AT 366 916 or AT 382 783.

[0085] According to a further aspect of the invention, the preparationaccording to the invention optionally contains a blood factor in theform of a zymogen or an active serine protease as a further component.Preferred further components are components having FEIB activity. Amongthem are, in particular, factor II, factor VII, factor IX, factor VIII,factor V and/or the active serine proteases thereof. Further componentscan also be phospholipids, Ca ions etc. According to a particularembodiment of the invention, the preparation according to the inventioncontains at least one further component having FEIB activity.

[0086] The preparation according to the invention can be provided as apharmaceutical preparation having factor Xa activity as a singlecomponent preparation or in combination with other factors as a multiplecomponent preparation.

[0087] Before processing to a pharmaceutical preparation, the purifiedprotein is subjected to the usual quality controls and brought into atherapeutically administrable form. In recombinant preparation, thepurified preparation is particularly tested for the absence of cellularand expression vector derived nucleic acids, preferably according to amethod as described in EP 0 714 987.

[0088] As, in principle, any biological material can be contaminatedwith infectious germs, the preparation is optionally treated forinactivation or depletion of viruses in order to produce a safepreparation.

[0089] A further aspect of the invention refers to the use of apreparation as described above in the preparation of a medicament. Amedicament containing a factor XΔ analogue according to the inventionand a correspondingly activated factor X analogue is particularly usefulin the treatment of patients suffering from blood coagulation disorderssuch as patients suffering from hemophilia or patients who havedeveloped inhibiting antibodies against the therapeutic agentadministered, e.g. against factor VIII or factor IX.

[0090] A further aspect of the invention relates to a method for thepreparation of the factor XΔ analogue and a preparation containing thefactor XΔ analogue according to the invention. The sequence encoding thefactor XΔ analogue is inserted into an appropriate expression system,and appropriate cells are transfected with the recombinant DNA.Preferably, permanent cell lines are established which express factor XΔanalogue. The cells are cultivated under optimal conditions for geneexpression, and factor X analogues are isolated either from a cellculture extract or from the cell culture supernatant. The recombinantmolecule can be further purified by all known chromatographic methods,such as anion or cation exchange, affinity or immunoaffinitychromatography or a combination thereof.

[0091] For the preparation of the factor XΔ analogues according to theinvention, the entire cDNA encoding the factor X is cloned in anexpression vector. This is effected according to generally known cloningtechniques. Subsequently, the nucleotide sequence encoding factor X ismodified such that the sequences encoding the amino acids Arg180 toArg234 are deleted and amino acids in the region between Gly173 andArg179, optionally Ile235, are modified such that a factor XΔ moleculeas described above can be produced. This is effected by geneticengineering techniques known from the state of the art, such as directedin vitro mutagenesis, deletion of sequences, e.g. by restrictiondigestion by endonucleases and insertion of other, changed sequences, orby PCR. The factor XΔ mutants thus prepared are then inserted into anexpression system appropriate for recombinant expression and areexpressed.

[0092] The factor XΔ analogues according to the invention can also beprepared by chemical synthesis.

[0093] The factor XΔ analogues are preferably produced by recombinantexpression. They can be prepared by means of genetic engineering withany usual expression systems, such as, for instance, permanent celllines or viral expression systems. Permanent cell lines are prepared bystable integration of the foreign DNA into the host cell chromosome of,e.g., vero, MRC5, CHO, BHK, 293, Sk-Hep1, particularly liver and kidneycells, or by an episomal vector derived, e.g., from the papilloma virus.Viral expression systems, such as, for instance, the vaccinia virus,baculovirus or retroviral systems, can also be employed. As cell lines,vero, MRC5, CHO, BHK, 293, Sk-Hep1, gland, liver and kidney cells aregenerally used. As eukaryotic expression systems, yeasts, endogenousglands (e.g. glands of transgenic animals) and other types of cells canbe used, too. Of course, transgenic animals can also be used for theexpression of the polypeptides according to the invention or derivativesthereof. For the expression of the recombinant proteins, CHO-DHFR⁻ cellshave proved particularly useful (Urlaub et al., Proc. Natl. Acad. Sci.,U.S.A., 77:4216-4220, 1980).

[0094] For the recombinant preparation of factor XΔ analogues accordingto the present invention, prokaryotic expression systems can be used,too. Systems allowing expression in E. coli or B. subtilis areparticularly useful.

[0095] The factor XΔ analogues are expressed in the respectiveexpression systems under control of a suitable promotor. For expressionin eukaryotes, all known promoters are suitable, such as SV40, CMV, RSV,HSV, EBV, β-actin, hGH or inducible promoters, such as, for instance,hsp or metallothionein promotor. The factor X analogues are preferablyexpressed under control of the β-actin promotor in CHO-DHFR⁻ cells.

[0096] According to an embodiment of the invention, the method forpreparing the preparation of the invention comprises the steps of:providing a DNA encoding a factor XΔ analogue, transforming a cell withthe recombinant DNA, expressing the factor X analogue, optionally in thepresence of a protease, isolating the factor X analogue, and optionalpurifying by means of a chromatographic method.

[0097] According to an embodiment of the process, the factor Xa analogueis directly isolated as a double chain molecule. A factor XΔ analoguehaving a modification allowing processing by a dibasic protease, such asfurin, is expressed in a cell, and the factor XΔ analogue is processedto double chain factor Xa analogue. The cell is preferably a cellexpressing a protease able to process, e.g. a dibasic protease, such asfurin or a derivative thereof. To improve or enhance processingefficiency, the cell can optionally be modified such that its proteaseexpression is enhanced. For instance, this can be effected byco-expression of a corresponding dibasic endoprotease, such asfurin/PACE, Kex2 or a derivative thereof. The factor XΔ analogueaccording to the invention can also be expressed in a cell having normalendogenous protease concentration, i.e. a suboptimal concentration forprocessing, resulting in incomplete processing into the double chainactive form. In this case, as long as single chain factor X analogue issecerned into the cell culture supernatant as described above,subsequent processing into factor Xa analogue is effected byco-cultivation with protease expressing cells or bringing into contactwith an optionally immobilized protease. The cell supernatant can alsobe pumped over a carrier matrix having protease bound thereto, thusyielding double chain factor Xa analogue in the eluate.

[0098] The factor Xa analogue thus obtained can subsequently beisolated, purified and optionally formulated as a pharmaceuticalcomposition and stored stably until further use, as described above. Thereaction conditions for the processing reaction and activation can beeasily optimized by a person skilled in the art according to theexperimental setup and the given basic conditions. For the contact time,the flow rate of the present reactants is of particular importance. Itshould be between 0.01 ml/min and 1 ml/min. Further important parametersare temperature, pH value and eluation conditions. After passage, factorXa analogue can optionally be further purified by selectivechromatography. It is particularly advantageous to conduct the processwith protease bound to a carrier, because when using a carrier,preferably chromatographic columns, the reaction setup allows anadditional purification step.

[0099] According to an embodiment, activation is effected by achromatographic step, wherein protease is immobilized on a carrier.Purified single chain factor XΔ analogue is conducted over a matrixhaving protease bound thereto, and purified factor Xa analogue isisolated from the eluate.

[0100] According to an aspect of the invention, a preparation containingactive factor Xa analogue is obtained by subjecting factor XΔ analogueprepared as described above to a processing/activation step and furtherprocessing the activated polypeptide to a purified preparationoptionally formulated as a pharmaceutical composition.

[0101] According to a further aspect of the production of a preparationcontaining single chain factor XΔ analogue, e.g., the factor XΔ analoguehaving a processing sequence for a dibasic protease is expressed in acell having endoprotease deficiency. The cell is preferably deficient ina dibasic endoprotease, such as kexin, furin, PACE or homologousderivatives thereof. From such an endoprotease deficient mutant cell,factor XΔ analogue can be isolated as a single chain molecule. Factor XΔanalogues having a processing site for a serine protease can beexpressed in any conventional cell, including furin positive cells, andisolated as a single chain molecule.

[0102] A factor X analogue thus isolated and optionally purified issubsequently brought into contact with a protease selected from thegroup of endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2,PC4, PACE 4, LPC/PC7, serine proteases, such as factor IIa, factor VIIa,factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or aderivative of these proteases, under conditions under which a singlechain factor X analogue is cleaved and activated to factor Xa analogue.

[0103] With the factor XΔ analogues according to the invention which areactivated by a process as described above to factor Xa analogues, apurified factor Xa analogue having high stability and structuralintegrity and being particularly free of inactive factor X/Xaintermediates is obtained.

[0104] The invention is described in more detail by the followingExamples and drawing figures, with the invention, however, not beingrestricted to these particular examplary embodiments.

[0105] Example 1 describes the construction and expression of rfactor X;Example 2 describes the processing of rfactor X into heavy and lightchain by furin; Example 3 describes the processing of pro-factor X bymeans of immobilized protease; Example 4 describes the activity ofrfactor X processed in vitro; Example 5 describes the expression ofrfactor X in furin deficient cells; Example 6 describes the constructionand expression of rfactor XΔ analogues; Example 7 describes thedetermination of N-termini of the factor X processing products; Example8 describes the expression and characterization of the FX deletionmutant having the site Arg-Val-Thr-Arg/Ile (rFXΔ^(RVTR/I)); Example 9describes in vitro activation of the protein rFXΔ^(RVTR/I) by r-furinderivatives.

[0106]FIG. 1 shows the nucleotide and amino acid sequence of factor X

[0107]FIG. 2 shows a schematic representation of the factor XΔ analogueshaving modified protease cleavage sites

[0108]FIG. 3 shows a schematic representation of the expression vectorphAct-rFX

[0109]FIG. 4 shows a Western blot analysis of rfactor X expressed in CHOcells before and after amplification

[0110]FIG. 5 shows a Western blot analysis of rfactor X after in vitrocleavage by furin derivatives

[0111]FIG. 6 shows a Western blot analysis of rfactor X moleculesexpressed in furin containing and furin deficient cells

[0112]FIG. 7 shows a schematic representation of rfactor XΔ analogueconstructs having modified C-termini of the heavy chain

[0113]FIG. 8 shows a schematic representation of the N-termini ofrfactor X processing products from CHO, CHO/r-furin and furin deficientcells

[0114]FIG. 9 shows a Western blot analysis of rfactor XΔ^(RVTR/I)expressed in CHO cells

[0115]FIG. 10 shows a Western blot analysis of rfactor XΔ^(RVTR/I) afterin vitro activation with furin derivative

[0116] The expression vectors were prepared by means of standard cloningtechniques (Maniatis et al., “Molecular Cloning”—A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., U.S.A., 1983).The preparation of DNA fragments by means of polymerase chain reaction(PCR) followed general methods (Clackson et al., 1991, PCR A practicalapproach. Ed. McPherson, Quirke, Taylor, p. 187-214).

EXAMPLE 1

[0117] Expression and Processing of Single Chain rFX to rFX Light/HeavyChain

[0118] a. Preparation of the rFX Expression Vector

[0119] For the preparation of recombinant FX (rFX), the cDNA of FX wasisolated from a human liver lambda-cDNA-library as described by Messieret al. (1991, Gene 99:291-294). A DNA fragment was amplified from apositive clone by means of PCR with oligonucleotide #2911(5′-ATTACTCGAGAAGCTTACCATGGGGCGCCCACTG-3′) (SEQ. ID No.1) as 5′-primerand oligonucleotide #2912 (5′-ATTACAATTGCTGCAGGGATCCAC-3′) (SEQ. ID. No.2) as 3′-primer, which DNA fragment contains the 1,467 kB FX codingsequence and 39 bp of the 3′-non-translated region, flanked by a XhoIcleavage site at the 5′-end and a MfeI cleavage site at the 3′-end. Inaddition, the sequence ACC was incorporated in front of the ATG of theFX by means of primer #2911 resulting in an optimal Kozak translationinitiation sequence. Subsequently, this PCR product was cloned asXhoI/MfeI fragment in the expression vector phAct cleaved with SalI andEcoRI. The resulting expression plasmid was designated as phAct-rFX(FIG. 3). The expression vector phAct comprises the humanbeta-actin-promotor, 78 bp 5′UTR and the intron, a multiple cloningcleavage site, and the SV40 polyadenylation site.

[0120] b. Expression of rFX in CHO Cells

[0121] In order to establish a stable rFX expressing cell line, dhfrdeficient CHO cells were co-transfected with the expression plasmidphAct-rFX and the selection marker plasmid pSV-dhfr. For all furtherexpression and function analyses, the cell cultures were incubated withserum free selection medium in the presence of 10 μg/ml vitamin K for 24hours. The expression of rFX in the resulting cell clones was detectedby means of the amount of antigen (ELISA, Asserachrom, BoehringerMannheim), and then the recombinant protein was characterized withSDS-PAGE (FIGS. 4A and B). As can be seen in the Western blot (FIG. 4A),in the initial clones and subclones thereof the recombinant FX proteinis present in the form of a light chain (LC) of 22 kD and a heavy chain(HC) of approximately 50 kD, which are identical with the plasmaticfactor X protein. In addition, a protein band is visible at 75 kD, whichcorresponds to the single chain (SC) molecule and the presence of whichin FX transfected CHO cells (Wolf et al., J. Biol. Chem.266:13726-13730, 1991) and in human plasma (Fair et al., Blood64:194-204, 1984) has been described. For the preparation of highlyexpressing clones, the initial clones were amplified with increasingamounts of methotrexate and subsequently subcloned to stabilization.Expression could be increased from about 200-500 ng/10 E6 cells or 1μg/ml, respectively, to 78 μg/10 E6 cells or 120 μg/ml, respectively,per 24 hours. Western blot analysis of these highly expressing cellclone supernatants (FIGS. 4B and 5A, lane 2) shows enrichment of thesingle chain rFX molecule and the presence of additional forms of thelight chain. Besides the 22 kD form of the light chain, whichcorresponds to the plasmatic form (completely carboxylated and withoutpropeptide) there are three further light chain variants of about 21 kD,22.5 kD, and 20 kD present. By means of N-terminal sequencing of therecombinant material, the heterogeneity of the light chain in theseclones was determined as a result of incomplete cleavage of thepropeptide (here: about 50% of the rFX material) and hypocarboxylation(here: about 50% of the rFX). The 21 kD protein is a hypocarboxylated,propeptide containing form, and the 20 kD protein is a hypocarboxylated,propeptide-free form of the light chain, while the 22.5 kD bandrepresents the fully carboxylated, but pro-peptide containing LC form.

EXAMPLE 2

[0122] Processing of Single Chain rFX in rFX Light/Heavy Chain byr-Furin Derivatives

[0123] Due to the similarity of the cleavage sites of factor Xpropeptide/N-terminus of the light chain (RVTR↓A) and of light/heavychain (RRKR↓S) to the furin consensus detection sequence (RXK/RR↓X), itwas possible to improve in vitro processing of single chain as well aspropeptide containing rFX molecules by r-furin derivatives. In theliterature, proteases are suspected for the two processing steps, which,however, are not furin (Rehemtulla et al., 1992, Blood 79:2349-2355;Wallin et al., 1994, Thromb. Res. 1994:395-403).

[0124] Cell culture supernatants of CHO-rFX and CHO-rfurin ΔTM6xHis(patent application EP 0 775 750) as well as CHO-rFX and non-transfectedCHO (as negative control) were mixed at a ratio of 1:1 and incubated at37° C. Aliquots of the reaction mixtures were tested for processed rFXbefore incubation (t=0) and after various incubation periods (t=2, 4, 6hours) by means of Western blot analysis (FIG. 5). The rFX was detectedin the cell culture supernatants by means of an anti-human FX antiserum(FIG. 5A) and a monoclonal antibody specific for the light chain of FX(FIG. 5B).

[0125] Contrary to the CHO-rFX/CHO mixture, CHO-rFX/CHO-rfurin showsalmost complete processing already after 2 hours of incubation at 37° C.(FIG. 5A, lane 7; FIG. 5B, lane 8). Single chain rFX is largely reactedto the light and heavy chain forms. In the area of the light chain, onlythe processed propeptide-free forms of 22 kD (carboxylated form) and 20kD (hypocarboxylated form) were found at a ratio of about 50:50. Byoptimizing cell culture conditions, this ratio can be improved in favorof the carboxylated form. Correct cleavage of the pro-sequence betweenArg-1 and Ala+1 and homogeneity of the N-terminus of the light chainwere determined by means of N-terminal sequencing. In the controlexperiment, wherein CHO-rFX was mixed with CHO-supernatants, no changein the rFX band pattern is visible even after 6 hours of incubation(FIG. 5A, lane 5; FIG. 5B, lane 6). This proves that r-furin in thesupernatant of CHO cells is biologically active and can process thepropeptide as well as the heavy/light chain of rFX.

EXAMPLE 3

[0126] Processing of Factor X by Means of Chelate-Tentacle GelImmobilized r-Furin

[0127] To determine whether a substrate can be cleaved by a column-boundr-furin derivative, a study was conducted as to whether in anexperimental setup Fractogel EMD® tentacle gel (Merck) can be usedinstead of Ni²⁺-NTA agarose as column matrix. As the metal ions arefarther apart from the actual column matrix than the Ni²⁺-NTA agarose,an improved sterical access to the bound r-furin derivative might beachieved. In the present setup, pro-factor X was processed by tentaclegel bound r-furin derivative:

[0128] Fractogel EMD® tentacle gel was loaded with Ni²+ions according tothe producer's instructions and equilibrated with fresh serum-free cellculture medium. Subsequently, the column was loaded with serum-freeCHO-r-furin derivative supernatant. Washing steps were carried out withserum-free cell culture medium containing increasing imidazoleconcentrations up to 40 mM. Then pro-factor X was passed over the columnas serum-free CHO supernatant. Processing of pro-factor X to doublechain factor X was detected in the effluent of the column by means ofWestern blot analysis with specific factor X antiserum.

EXAMPLE 4

[0129] Activity of Recombinant Factor X Processed in vitro

[0130] Recombinant factor X precursor was incubated with and withoutr-furin at 4° C. At different times, samples were taken and frozen at−20° C. After the incubation was completed (after 4 days), all sampleswere tested for FX activity using a FX Coatest Kit (Chromogenix). 50 μlof each supernatant were mixed with 50 μl FX deficient human plasma, andrFX was reacted with snake venom (RVV) to rFXa in the presence of CaCl₂according to the producer's instructions; rFXa then hydrolyzes thechromogenic substrate (S-2337) and leads to the release ofyellow-coloured paranitroaniline. As the amount of rFXa and theintensity of the colour are proportionate to each other, the amount ofrFX/ml cell culture supernatant which can be activated to rFXa can bedetermined by means of a calibration line interpolated from values of aplasma dilution series. Using these results and the known amount of rFXantigen (ELISA data), the proportion of rfactor X activated to factor Xacan be calculated in %. The results are presented in table 1.

[0131] In order to exclude nonspecific, proteolytic activity in CHO andCHO-r-furin supernatants, the mixture of these two cell culturesupernatants was tested, too.

[0132] Even after 4 days, CHO-rFX incubated with CHO supernatants(without r-furin) as control displayed no substantial change in rFXaactivity, which was about 800 mU/ml and corresponded to 50% to 60% offunctional rFX due to experimental variations. When, in comparison,CHO-rFX was incubated with CHO-r-furin, rFX activity increased steadilyduring incubation, rising from about 60% (T=0) to 86% (table 1). Thisproves that in vitro processing of CHO-rFX from highly expressing clonesusing r-furin derivative substantially improves the proportion of rFXthat can be activated to functional rFXa. TABLE 1 Amount of functionalincubation activity antigen portion of (days) (mU) (μg/ml) rFX (%)CHO-rFX + 0 814 14 58 CHO 1 847 14 61 2 835 14 60 3 790 14 56 4 763 1455 CHO-rFX + 0 853 14 61 CHO-rFurin 1 1018 14 73 2 1099 14 79 3 1135 1481 4 1198 14 86 CHO + 0 CHO-rFurin Plasma FX 585 500 mU

EXAMPLE 5

[0133] Expression of Recombinant Factor X in Furin Deficient Cells

[0134] As shown in the previous Examples, in the case of factor Xprecursor protein, furin mediates propeptide cleavage as well ascleavage of the single chain to light/heavy chain in vitro. Thissuggests that these steps are also effected endogenously in the cell byubiquitous furin with varying efficiency depending on the amount ofexpressed rfactor X. This in turn leads to the production of a mixtureof heterogenous rfactor X forms.

[0135] One way to prepare a form of rfactor X molecules which is ashomogeneous as possible and also stable is to prevent cleavage ofrfactor X by endogenous proteases, particularly furin, and thus toproduce functionally inactive rfactor X precursors (which can betransformed into its functionally active form later by means ofdownstream processing, ideally directly before use). This process willbe particularly useful in the preparation of FX deletion mutantscontaining a furin cleavage site instead of the original activationsite. In these constructs, such a recombinant rFX mutant in vivo can beactivated by endogenous furin and lead to the secretion of activated,more instable rFX forms. Degradation of these forms by CHO proteases,e.g. under cell culture conditions of high cell lysis, during storage ofthe cell culture supernatants or the purifying process could result ininactive degradation products (Wolf et al., 1991).

[0136] This aim can, for instance, be achieved by supplementing the cellculture medium with agents which can reduce or prevent intracellularfurin activity.

[0137] Another way is to use cells which are furin deficient a priori(Möhring et al., 1983, Infect. Immun. 41:998-1009; Ohnishi et al., 1994,J. Virol. 68:4075-4079; Gordon et al., 1995, Infect. Immun. 63:82-87).

[0138] For this purpose, a furin deficient CHO cell clone FD11 (Gordonet al., 1995, Infect. Immun. 63:82-87) was co-transfected with 20 μgphAct-FX and 1 μg pUCSV-neo (containing the neomycin resistance gene inthe pUC vector under control of the SV40 promotor). In order to obtainstable clones, the medium was supplemented with 0.8 μg G418/ml.Comparing secerned rfactor X molecules in serum free supernatants of afurin containing and a furin deficient CHO clone, Western blot showsthat rfactor X precursor is not processed in the furin deficient cellsand only single chain factor X precursor is present (FIG. 6); incontrast, rfactor X is still completely processed by “normal” cells withmodest expression, but is processed only to a very limited extent withhigher expression in spite of endogenous furin. Due to the low degree ofrFX expression of the cell clone used, the light chain of rfactor X hereis not visible in the blot.

EXAMPLE 6

[0139] Preparation of Factor XΔ Analogues (at Present, the ApplicantRegards this as the Best Mode for Carrying Out the Invention)

[0140] 6.1. Construction of Expression Plasmids for the Preparation ofFX Deletion Mutants

[0141] Factor X deletion mutants differ from the factor X wild typesequence in the deletion of the app. 4.5 kDa activation peptides betweenamino acid 180 and 234. In addition, various cleavage sites wereintroduced into the C-terminus of the light chain and/or the N-terminusof the heavy chain by means of mutagenesis, which sites function toactivate the single chain factor X molecule resulting therefrom to theactivated polypeptide. Expression plasmids for these factor X deletionmutants are all derived from phAct-FX (described in Example 1).

[0142] In order to simplify the cloning of factor X deletion mutants,the HindIII-NaeI DNA fragment from plasmid phAct-FX, which comprises thefactor X encoding region from position +1 to +1116, was inserted intothe HindIII/SmaI restriction cleavage sites of plasmid pUC19. Theresulting plasmid was designated as pUC/FX. In order to delete theactivation peptide and to incorporate new cleavage sites, e.g. furin,FXIa, FXIIa, FXa, FIIa cleavage sites, the Bsp120I/BstXI FX DNA fragmentfrom the pUC/FX vector was replaced by synthetic oligonucleotides. Inorder to incorporate a thrombin or FXIa cleavage site, theBstXI-3′-overlap was smoothened by mung bean nuclease, so that aminoacid Ile at position 235 could be exchanged, too. Subsequently, thedeleted factor X DNA fragments were cloned in plasmid PACT-FX viaHindIII-AgeI.

[0143] In order to prepare the Asp-Phe-Thr-Arg/Val FXIa cleavage site,the oligonucleotide sense #0009 (5′-GG CCC TAC CCC TGT GGG AAA CAG GACTTC ACC AGG GTG-31) (SEQ. ID. No. 3) and the oligonucleotide antisense#0010 (5′-CAC CCT GGT GAA GTC CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID.No. 4) were used and inserted into the Bsp120I and the mung beannuclease treated BstXI sites. Thus, the amino acids from position 176 to178 and 235 were mutated into Asp-Phe-Thr and Val (FIG. 2A).

[0144] In order to prepare the Arg/Thr fIIa cleavage site, theoligonucleotide sense #0011 (5′-GG CCC TAC CCC TGT GGG AAA CAG ACC CTGGAA CGG ACC-3′) (SEQ. ID. No. 5) and the oligonucleotide antisense #0012(5′-GGT CCG TTC CAG GGT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 6)were used and inserted into the Bsp120I and the mung bean nucleasetreated BstXI sites. Thus, the amino acid Ile at position 235 wasmutated into Thr (FIG. 2B).

[0145] In order to prepare the Ile-Lys-Pro-Arg/Ile FXIIa cleavage site,the oligonucleotide sense #0013 (5′-GG CCC TAC CCC TGT GGG AAA CAG ATCAAG CCC AGG ATC-3′) (SEQ. ID. No. 7) and the oligonucleotide antisense#0014 (5′-CT GGG CTT GAT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 8)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of position 176 to 178 were mutated into Ile-Lys-Pro (FIG. 2C).

[0146] In order to prepare the Ser-Met-Thr-Arg/Ile kallikrein cleavagesite, the oligonucleotide sense #0015 (5′-GG CCC TAC CCC TGT GGG AAA CAGAGC ATG ACC AGG ATC-3′) (SEQ. ID. No. 9) and the oligonucleotide #0016(5′-CT GGT CAT GCT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 10) wereused and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of position 176 to 178 were mutated into Ser-Met-Thr (FIG. 2D).

[0147] In order to prepare a Met-Lys-Thr-Arg/Ile FXa cleavage site, theoligonucleotide sense #0033 (5′-GG CCC TAC CCC TGT GGG AAA CAG ATG AAAACG AGG ATC-3′) (SEQ. ID. No. 11) and the oligonucleotide antisense#0034 (5′-CT CGT TTT CAT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 12)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of position 176 to 178 were mutated from Thr-Leu-Glu intoMet-Lys-Thr (FIG. 2E).

[0148] In order to prepare an. Ile-Glu-Gly-Arg/Ile FXa cleavage site,the oligonucleotide sense #0035 (5′-GG CCC TAC CCC TGT GGG AAA CAG ATCGAG GGA AGG ATC-3′) (SEQ. ID. No. 13) and the oligonucleotide antisense#0036 (5′-CT TCC CTC GAT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 14)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids in position 176 to 178 were mutated from Thr-Leu-Glu intoIle-Glu-Gly (FIG. 2F).

[0149] In order to prepare an Arg-Arg-Lys-Arg/Ile furin cleavage site,the oligonucleotide sense #0017 (5′-GG CCC TAC CCC TGT GGG AAA CAG AGGAGG AAG AGG ATC-3′) (SEQ. ID. No. 15) and the oligonucleotide antisense#0018 (5′-CT CTT CCT CCT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 16)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids in positions 176 to 178 were mutated into Arg-Arg-Lys (FIG. 2G).

[0150] In order to prepare an Arg-Val-Arg-Arg/Ile furin cleavage site,the oligonucleotide sense #0019 (5′-GG CCC TAC CCC TGT GGG AAA CAG AGGGTG AGG AGG ATC-3′) (SEQ. ID. No. 17) and the oligonucleotide antisense#0020 (5′-CT CCT CAC CCT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 18)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of positions 176 to 178 were mutated into Arg-Val-Arg (FIG. 2G).

[0151] In order to prepare an Arg-Arg-Arg-Arg/Ile furin cleavage site,the oligonucleotide sense #0021 (5′-GG CCC TAC CCC TGT GGG AAA CAG AGGAGG AGG AGG ATC-3′) (SEQ. ID. No. 19) and the oligonucleotide antisense#0022 (5′-CT CCT CCT CCT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 20)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of positions 176 to 178 were mutated into Arg-Arg-Arg (FIG. 2G).

[0152] In order to prepare an Arg-Pro-Lys-Arg/Ile furin cleavage site,the oligonucleotide sense #0023 (5′-GG CCC TAC CCC TGT GGG AAA CAG AGGCCC AAG AGG ATC-3′) (SEQ. ID. No. 21) and the oligonucleotide antisense#0024 (5′-CT CTT GGG CCT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 22)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of positions 176 to 178 were mutated into Arg-Pro-Lys (FIG. 2G).

[0153] In order to prepare an Ile-Arg-Lys-Arg/Ile furin cleavage site,the oligonucleotide sense #0025 (5′-GG CCC TAC CCC TGT GGG AAA CAG ATCAGG AAG AGG ATC-3′) (SEQ. ID. No. 23) and the oligonucleotide antisense#0026 (5′-CT CTT CCT GAT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 24)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of positions 176 to 178 were mutated into Ile-Arg-Lys (FIG. 2G).

[0154] In order to prepare an Arg-Ser-Lys-Arg/Ile furin cleavage site,the oligonucleotide sense #0027 (5′-GG CCC TAC CCC TGT GGG AAA CAG AGGAGC AAG AGG ATC-3′) (SEQ. ID. No. 25) and the oligonucleotide antisense#0028 (5′-CT CTT GCT CCT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 26)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of positions 176 to 178 were mutated into Arg-Ser-Lys (FIG. 2G).

[0155] In order to prepare an Arg-Val-Thr-Arg/Ile furin cleavage site,the oligonucleotide sense #0029 (5-GG CCC TAC CCC TGT GGG AAA CAG AGGGTC ACG AGG ATC-3′) (SEQ. ID. No. 27) and the oligonucleotide antisense#0030 (5′-CT CGT GAC CCT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 28)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of positions 176 to 178 were mutated into Arg-Val-Thr (FIG. 2G).

[0156] In order to prepare an Arg-Leu-Lys-Arg/Ile furin cleavage site,the oligonucleotide sense #0031 (5′-GG CCC TAC CCC TGT GGG AAA CAG AGGCTG AAA AGG ATC-3′) (SEQ. ID. No. 29) and the oligonucleotide antisense#0032 (5′-CT TTT CAG CCT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 30)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of positions 176 and 178 were mutated into Arg and Lys (FIG. 2G).

[0157] In order to prepare an Pro-Gln-Gly-Arg/Ile FXa cleavage site, theoligonucleotide sense #0037 (5′-GG CCC TAC CCC TGT GGG AAA CAG CCC CAAGGA AGG ATC-3′) (SEQ. ID. No. 31) and the oligonucleotide antisense#0038 (5′-CT TCC TTG GGG CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 32)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids in positions 176 to 178 were mutated from Thr-Leu-Glu intoPro-Gln-Gly (FIG. 2H).

[0158] In order to prepare the Thr-Ser-Thr-Arg/Ile FXIIa cleavage site,the oligonucleotide sense #0039 (5′-GG CCC TAC CCC TGT GGG AAA CAG ACGAGC ACG AGG ATC-3′) (SEQ. ID. No. 33) and the oligonucleotide antisense#0040 (5′-CT CGT GCT CGT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 34)were used and inserted into the Bsp120I and BstXI sites. Thus, the aminoacids of positions 176 and 178 were mutated into Ser-Thr (FIG. 2I).

[0159] In order to prepare an Arg/Ile trypsin cleavage site, theoligonucleotide #0041 (5′-GG CCC TAC CCC TGT GGG AAA CAG ACC CTG GAA CGGATC-3′) (SEQ. ID. No. 35) and the oligonucleotide antisense #0042 (5′-CGTTC CAG GGT CTG TTT CCC ACA GGG GTA G-3′) (SEQ. ID. No. 36) were usedand inserted into the Bsp120I and BstXI sites (FIG. 2J).

[0160] The resulting expression plasmids (see FIG. 3) comprise the humanbeta-actin-promotor, 78 bp of 5′UTR, the beta-actin-intron, the modifiedfactor X sequence, and 39 bp of the 3′UTR and the SV40 polyadenylationsite.

[0161] 6.2. Construction of Expression Plasmids for the Preparation ofFXβ Analogue

[0162] These constructs were derived from the factor XΔ analogueconstructs described above by introducing a TGA stop codon into position470. The amino acids from position 457 to the stop codon were removed bySpeI and partial BstEII digestion and replaced by the oligonucleotidepair #0003 (5′-GTC ACC GCC TTC CTC AAG TGG ATC GAC AGG TCC ATG AAA ACCAGG TGA A-3¹) (SEQ. ID. No. 37) and #0004 (5′-CTA GTT CAC CTG GTT TTCATG GAC CTG TCG ATC CAC TTG AGG AAG GCG-3′) (SEQ. ID. No. 38). FIG. 7 isa schematic representation of the factor XΔβ analogue constructs. Inorder to simplify the Figure, all factor XΔβ analogues are representedas a general construct wherein the variable amino acids in the cleavagesite region are designated as a shaded “X”.

[0163] 6.3. Construction of Expression Plasmids for the Production ofFXΔα Analogue

[0164] By activating factor X by cleaving off the 4.5 kDa activationpeptide at the N-terminal end of the heavy chain, the factor Xaα form isgenerated. This form is subsequently reacted to the FXaβ form byautoproteolytic activity and cleavage of the C-terminus of the heavychain between Arg469 and Gly470. For the preparation of factor Xexpression plasmids leading to the production of factor XΔ analogues,which will be present after activation exclusively in the FXaα formhaving intact β-peptide, the amino acid Arg469 was mutated to Lys sothat the C-terminal region of the heavy chain can not be processed anymore.

[0165] For this purpose, the DNA sequence of factor X encoding theC-terminal amino acid sequence was removed from position 1363 to thestop signal by partial BstEII-SpeI digestion and replaced by two ligatedoligonucleotide pairs. Oligonucleotide #0005 (5′-GTC ACC GCC TTC CTC AAGTGG ATC GAC AGG TCC ATG AAA ACC AAG GGC TTG CCC AAG-3¹) (SEQ. ID. No.39) and oligonucleotide #0006 (5′-TTG GCC TTG GGC AAG CCC TTG GTT TTCATG GAC CTG TCG ATC CAC TTG AGG AAG GCG-3′) (SEQ. ID. No. 40) wereligated with oligonucleotide #0007 (5′-GCC AAG AGC CAT GCC CCG GAG GTCATA ACG TCC TCT CCA TTA AAG TGA GAT CCC A-3′) (SEQ. ID. No. 41) andoligonucleotide #0008 (5′-CTA GTG GGA TCT CAC TTT AAT GGA GAG GAC GTTATG ACC TCC GGG GCA TGG CTC-3′) (SEQ. ID. No. 42). The mutation of aminoacid Arg469 is introduced by the oligonucleotide pair #0005-#0006. FIG.7 is a schematic representation of the FXΔ analogues.

EXAMPLE 7

[0166] Determination of the N-Termini of Factor X and ProcessingProducts With and Without r-Furin

[0167] Recombinant factor X was expressed in CHO cells having endogenousfurin, as described in Example 1, and in furin deficient cells, asdescribed in Example 5. rFactor X was isolated from cell culturesupernatant of highly expressing CHO-rFX clones, which was a) notpre-treated, b) incubated at 37° C. for 12 hours and c) pre-treated withCHO-r-furin supernatant at 37° C. for 12 hours, as well as from cellculture supernatant of CHO-FD11-rFX clones which was d) not pre-treatedand e) pre-treated with CHO-r-furin supernatant at 37° C. for 12 hours.The terminal N-terminal amino acids of factor X and the processingproducts of the individual reaction mixtures a) to e) were determined byEdman analysis. FIG. 8 is a schematic representation of the results.

[0168] rFactor X from highly expressing CHO cells is present in the formof the mature heavy and light chains as well as in the single chainform, partly still containing propeptide. After incubation of these cellculture supernatants for 12 hours at 37° C. (b), additional faultyN-termini of the rFX light chain having 3 additional amino acidsVal38-Thr39-Arg40 are formed, as described by Wolf et al. (1991, J. Bio.Chem. 266:13726-13730). These cryptic ends are also found whensequencing rFX material from non-pre-treated CHO-FD11 cells (d). Thisobservation shows that the formation of these faulty N-termini can beprevented by reasonable conditions, i.e. cell culture conditions,storage and purifying processes in order to minimize rFX proteolysis byCHO proteases.

[0169] Contrary to the purified material from CHO cells (a and b), rFXfrom non-amplified, furin deficient cells (d) is only present in theform of unprocessed single chain precursors N-terminal sequencescorresponding to the propeptide portion are not found, either. Thisshows that single chain rFX precursor is not processed any more tolight/heavy chain in furin deficient CHO cells (d), which suggests acentral role of the endoprotease furin in this processing step in vivo.In addition, it shows that rFX molecules containing propeptide are alsoprocessed in furin deficient CHO cells, i.e. that furin does not play anessential role in this processing step in vivo. After incubation of rFXfrom CHO cells (c) and CHO-FD11 cells (e) in the presence of furin, onlylight and heavy chains having correct N-termini are found. This provesthat the single chain FX precursors as well as the propeptide containingrFX molecules are reacted to homogenous, mature factor X by in vitroprocessing. Thus, factor X processed in the presence of furin exhibitsexceptional structural integrity.

EXAMPLE 8

[0170] Expression and Characterization of the Recombinant FX DeletionMutant Having the Cleavage Site Arg-Val-Thr-Arg/Ile (FXΔ^(RVTR/I))

[0171] The expression plasmid encoding the FX deletion mutant having thecleavage site Arg-Val-Thr-Arg/Ile (FXΔ^(RVTR/I)) was co-transfected withthe selection marker pSV/dhfr in dhfr deficient CHO cells as describedin Example 1. The recombinant protein FXΔ^(RVTR/I) from permanent CHOclones was characterized by means of Western blot analysis. As can beseen in. FIG. 9, lane 4, the recombinant protein is present in the formof a double band of approximately 56 and 50 kD. No FX reactive materialis detectable in the cell culture supernatant of non-transfected CHOcells (lane 2). According to these results, it is impossible that theseprotein bands result from impurities of the analyzed supernatants ofwild type FX from the residues of bovine serum in the cell culturemedium. Therefore, the double band is possibly caused by differentpost-translational modifications, e.g. the presence of the propeptide ordifferent glycosylation of the rFXΔ^(RVTR/I) molecule.

[0172] The cleavage site Arg-Val-Thr-Arg/Ile inserted into thisconstruct is identical with the propeptide cleavage site of the wildtype FX molecule, which is efficiently recognized and cleaved in vivo bya CHO endoprotease (see Example 7). The Western blot analysis shows noadditional 35 kD and 31 kD heavy FX molecules, which would correspond tothe activated α- and β-forms of the rFXΔ^(RVTR/I) heavy chain. Theseresults show that either the amount of endoprotease is not sufficient toactivate the protein or/and that the cleavage site Arg-Val-Thr-Arg/Ileis not or not effectively recognized and cleaved in vivo in the presentsequence environment. Consequently, rFXΔ^(RVTR/I) is practically onlypresent in the single chain form.

EXAMPLE 9

[0173] Activation of the Recombinant rFXΔ^(RVTR/I) Protein by Means ofRecombinant Furin Derivatives in vitro

[0174] Although the cleavage site Arg-Val-Thr-Arg in the rFX propeptideis recognized in vivo by a protease other than furin, Example 2 provesthat this sequence is cleaved very efficiently and correctly by anr-furin derivative in vitro.

[0175] Mixing experiments were carried out in order to test the abilityof rFXΔ^(RVTR/I) protein to be activated by r-furin in vitro. Cellculture supernatant from CHO-FXΔ^(RVTR/I) cells were mixed with purifiedr-furin derivative r-furinΔCys-spacer-10xHis (see patent applicationEP-0 775 750-A2) in the presence of 20 mM Hepes pH 7.0, 150 mM NaCl, 4mM CaCl₂ and 0.1% BSA at a ratio of 1:1. In a control experiment, theCHO-rFXΔ^(RVTR/I) supernatant was mixed only with BSA containing bufferat the same ratio. The addition of BSA is meant to stabilize theenzymatic activity of the r-furin derivative and the activatedrFXΔ^(RVTR/I) products consequently formed. Aliquots of the reactionmixture were tested before and after an incubation period of 6, 24, 48and 72 hours (t=O, t=6, t=24, t=48, t=72) at 37° C. for rFXΔ^(RVTR/I)processing by means of Western blot analysis (FIG. 10). In the mixingexperiment without r-furin addition (FIG. 10B), no change in the bandpattern is visible during the incubation period (lanes 4 to 9). Due tothe presence of BSA in the reaction mixtures, only the lighterrFXΔ^(RVTR/I) molecules (50 kD) are easily visible, because the 56 kDheavy molecules are covered by the BSA band. In the presence of ther-furin derivative (FIG. 10A), a 35 kD protein band appears alreadyafter 6 hours of incubation (lane 5), which corresponds to the a-form ofthe FX heavy chain (cf. lane 9). This protein accumulates in the courseof incubation and is subsequently reacted to the proteolytic β-form, asalready known in the case of plasma FX, which β-form forms byproteolytic conversion from the α-form (lanes 7 and 8). Light chains of22 kD and 20 kD appear parallel to the detection of the activated formsof the heavy chains, which light chains were identified as propeptidefree, carboxylated LC2 form (corresponding to the actually functionalform) or as propeptide free, hypocarboxylated LC4 form of the lightchain in Example 1.b. The presence of the hypocarboxylated LC4 formproves that the post-translational modification mechanisms are limitedin the analysed CHO clones. Although the 50 kD bond appears to beunchanged, while apparently the 56 kd form is directly degraded tolight/heavy chains, in fact the 56 kD molecule at first is convertedinto the 50 kD form, and only subsequently is cleaved into a light and aheavy chain. This is due to the presence of the propeptide in the. 56 kDmolecule which at first is removed by forming the 50 kD form.

[0176] This proves that the rFXΔ^(RVTR/I) construct can be activated invitro by r-furin derivatives via an inserted Arg-Val-Thr-Arg/Ilecleavage site and the resulting processing products of the rFXΔ^(RVTR/I)construct correspond to those of plasma FXa in size. The emergence ofFXΔβ, which is formed due to autoproteolytic processing of FXΔα, showsthe functionality of the rFXΔ^(RVTR/I) molecule.

1 145 34 base pairs nucleic acid single linear DNA 1 ATTACTCGAGAAGCTTACCA TGGGGCGCCC ACTG 34 24 base pairs nucleic acid single linearDNA 2 ATTACAATTG CTGCAGGGAT CCAC 24 38 base pairs nucleic acid singlelinear DNA 3 GGCCCTACCC CTGTGGGAAA CAGGACTTCA CCAGGGTG 38 34 base pairsnucleic acid single linear DNA 4 CACCCTGGTG AAGTCCTGTT TCCCACAGGG GTAG34 38 base pairs nucleic acid single linear DNA 5 GGCCCTACCC CTGTGGGAAACAGACCCTGG AACGGACC 38 34 base pairs nucleic acid single linear DNA 6GGTCCGTTCC AGGGTCTGTT TCCCACAGGG GTAG 34 38 base pairs nucleic acidsingle linear DNA 7 GGCCCTACCC CTGTGGGAAA CAGATCAAGC CCAGGATC 38 30 basepairs nucleic acid single linear DNA 8 CTGGGCTTGA TCTGTTTCCC ACAGGGGTAG30 38 base pairs nucleic acid single linear DNA 9 GGCCCTACCC CTGTGGGAAACAGAGCATGA CCAGGATC 38 30 base pairs nucleic acid single linear DNA 10CTGGTCATGC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 11 GGCCCTACCC CTGTGGGAAA CAGATGAAAA CGAGGATC 38 30 base pairsnucleic acid single linear DNA 12 CTCGTTTTCA TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 13 GGCCCTACCC CTGTGGGAAACAGATCGAGG GAAGGATC 38 30 base pairs nucleic acid single linear DNA 14CTTCCCTCGA TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 15 GGCCCTACCC CTGTGGGAAA CAGAGGAGGA AGAGGATC 38 30 base pairsnucleic acid single linear DNA 16 CTCTTCCTCC TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 17 GGCCCTACCC CTGTGGGAAACAGAGGGTGA GGAGGATC 38 30 base pairs nucleic acid single linear DNA 18CTCCTCACCC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 19 GGCCCTACCC CTGTGGGAAA CAGAGGAGGA GGAGGATC 38 30 base pairsnucleic acid single linear DNA 20 CTCCTCCTCC TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 21 GGCCCTACCC CTGTGGGAAACAGAGGCCCA AGAGGATC 38 30 base pairs nucleic acid single linear DNA 22CTCTTGGGCC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 23 GGCCCTACCC CTGTGGGAAA CAGATCAGGA AGAGGATC 38 30 base pairsnucleic acid single linear DNA 24 CTCTTCCTGA TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 25 GGCCCTACCC CTGTGGGAAACAGAGGAGCA AGAGGATC 38 30 base pairs nucleic acid single linear DNA 26CTCTTGCTCC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 27 GGCCCTACCC CTGTGGGAAA CAGAGGGTCA CGAGGATC 38 30 base pairsnucleic acid single linear DNA 28 CTCGTGACCC TCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 29 GGCCCTACCC CTGTGGGAAACAGAGGCTGA AAAGGATC 38 30 base pairs nucleic acid single linear DNA 30CTTTTCAGCC TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 31 GGCCCTACCC CTGTGGGAAA CAGCCCCAAG GAAGGATC 38 30 base pairsnucleic acid single linear DNA 32 CTTCCTTGGG GCTGTTTCCC ACAGGGGTAG 30 38base pairs nucleic acid single linear DNA 33 GGCCCTACCC CTGTGGGAAACAGACGAGCA CGAGGATC 38 30 base pairs nucleic acid single linear DNA 34CTCGTGCTCG TCTGTTTCCC ACAGGGGTAG 30 38 base pairs nucleic acid singlelinear DNA 35 GGCCCTACCC CTGTGGGAAA CAGACCCTGG AACGGATC 38 30 base pairsnucleic acid single linear DNA 36 CGTTCCAGGG TCTGTTTCCC ACAGGGGTAG 30 49base pairs nucleic acid single linear DNA 37 GTCACCGCCT TCCTCAAGTGGATCGACAGG TCCATGAAAA CCAGGTGAA 49 48 base pairs nucleic acid singlelinear DNA 38 CTAGTTCACC TGGTTTTCAT GGACCTGTCG ATCCACTTGA GGAAGGCG 48 57base pairs nucleic acid single linear DNA 39 GTCACCGCCT TCCTCAAGTGGATCGACAGG TCCATGAAAA CCAAGGGCTT GCCCAAG 57 57 base pairs nucleic acidsingle linear DNA 40 TTGGCCTTGG GCAAGCCCTT GGTTTTCATG GACCTGTCGATCCACTTGAG GAAGGCG 57 55 base pairs nucleic acid single linear DNA 41GCCAAGAGCC ATGCCCCGGA GGTCATAACG TCCTCTCCAT TAAAGTGAGA TCCCA 55 54 basepairs nucleic acid single linear DNA 42 CTAGTGGGAT CTCACTTTAA TGGAGAGGACGTTATGACCT CCGGGGCATG GCTC 54 1467 base pairs nucleic acid single linearcDNA CDS 1...1467 Factor X 43 ATG GGG CGC CCA CTG CAC CTC GTC CTG CTCAGT GCC TCC CTG GCT GGC 48 Met Gly Arg Pro Leu His Leu Val Leu Leu SerAla Ser Leu Ala Gly 1 5 10 15 CTC CTG CTG CTC GGG GAA AGT CTG TTC ATCCGC AGG GAG CAG GCC AAC 96 Leu Leu Leu Leu Gly Glu Ser Leu Phe Ile ArgArg Glu Gln Ala Asn 20 25 30 AAC ATC CTG GCG AGG GTC ACG AGG GCC AAT TCCTTT CTT GAA GAG ATG 144 Asn Ile Leu Ala Arg Val Thr Arg Ala Asn Ser PheLeu Glu Glu Met 35 40 45 AAG AAA GGA CAC CTC GAA AGA GAG TGC ATG GAA GAGACC TGC TCA TAC 192 Lys Lys Gly His Leu Glu Arg Glu Cys Met Glu Glu ThrCys Ser Tyr 50 55 60 GAA GAG GCC CGC GAG GTC TTT GAG GAC AGC GAC AAG ACGAAT GAA TTC 240 Glu Glu Ala Arg Glu Val Phe Glu Asp Ser Asp Lys Thr AsnGlu Phe 65 70 75 80 TGG AAT AAA TAC AAA GAT GGC GAC CAG TGT GAG ACC AGTCCT TGC CAG 288 Trp Asn Lys Tyr Lys Asp Gly Asp Gln Cys Glu Thr Ser ProCys Gln 85 90 95 AAC CAG GGC AAA TGT AAA GAC GGC CTC GGG GAA TAC ACC TGCACC TGT 336 Asn Gln Gly Lys Cys Lys Asp Gly Leu Gly Glu Tyr Thr Cys ThrCys 100 105 110 TTA GAA GGA TTC GAA GGC AAA AAC TGT GAA TTA TTC ACA CGGAAG CTC 384 Leu Glu Gly Phe Glu Gly Lys Asn Cys Glu Leu Phe Thr Arg LysLeu 115 120 125 TGC AGC CTG GAC AAC GGG GAC TGT GAC CAG TTC TGC CAC GAGGAA CAG 432 Cys Ser Leu Asp Asn Gly Asp Cys Asp Gln Phe Cys His Glu GluGln 130 135 140 AAC TCT GTG GTG TGC TCC TGC GCC CGC GGG TAC ACC CTG GCTGAC AAC 480 Asn Ser Val Val Cys Ser Cys Ala Arg Gly Tyr Thr Leu Ala AspAsn 145 150 155 160 GGC AAG GCC TGC ATT CCC ACA GGG CCC TAC CCC TGT GGGAAA CAG ACC 528 Gly Lys Ala Cys Ile Pro Thr Gly Pro Tyr Pro Cys Gly LysGln Thr 165 170 175 CTG GAA CGC AGG AAG AGG TCA GTG GCC CAG GCC ACC AGCAGC AGC GGG 576 Leu Glu Arg Arg Lys Arg Ser Val Ala Gln Ala Thr Ser SerSer Gly 180 185 190 GAG GCC CCT GAC AGC ATC ACA TGG AAG CCA TAT GAT GCAGCC GAC CTG 624 Glu Ala Pro Asp Ser Ile Thr Trp Lys Pro Tyr Asp Ala AlaAsp Leu 195 200 205 GAC CCC ACC GAG AAC CCC TTC GAC CTG CTT GAC TTC AACCAG ACG CAG 672 Asp Pro Thr Glu Asn Pro Phe Asp Leu Leu Asp Phe Asn GlnThr Gln 210 215 220 CCT GAG AGG GGC GAC AAC AAC CTC ACC AGG ATC GTG GGAGGC CAG GAA 720 Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg Ile Val Gly GlyGln Glu 225 230 235 240 TGC AAG GAC GGG GAG TGT CCC TGG CAG GCC CTG CTCATC AAT GAG GAA 768 Cys Lys Asp Gly Glu Cys Pro Trp Gln Ala Leu Leu IleAsn Glu Glu 245 250 255 AAC GAG GGT TTC TGT GGT GGA ACT ATT CTG AGC GAGTTC TAC ATC CTA 816 Asn Glu Gly Phe Cys Gly Gly Thr Ile Leu Ser Glu PheTyr Ile Leu 260 265 270 ACG GCA GCC CAC TGT CTC TAC CAA GCC AAG AGA TTCAAG GTG AGG GTA 864 Thr Ala Ala His Cys Leu Tyr Gln Ala Lys Arg Phe LysVal Arg Val 275 280 285 GGG GAC CGG AAC ACG GAG CAG GAG GAG GGC GGT GAGGCG GTG CAC GAG 912 Gly Asp Arg Asn Thr Glu Gln Glu Glu Gly Gly Glu AlaVal His Glu 290 295 300 GTG GAG GTG GTC ATC AAG CAC AAC CGG TTC ACA AAGGAG ACC TAT GAC 960 Val Glu Val Val Ile Lys His Asn Arg Phe Thr Lys GluThr Tyr Asp 305 310 315 320 TTC GAC ATC GCC GTG CTC CGG CTC AAG ACC CCCATC ACC TTC CGC AT 1008 Phe Asp Ile Ala Val Leu Arg Leu Lys Thr Pro IleThr Phe Arg Met 325 330 335 AAC GTG GCG CCT GCC TGC CTC CCC GAG CGT GACTGG GCC GAG TCC AC 1056 Asn Val Ala Pro Ala Cys Leu Pro Glu Arg Asp TrpAla Glu Ser Thr 340 345 350 CTG ATG ACG CAG AAG ACG GGG ATT GTG AGC GGCTTC GGG CGC ACC CA 1104 Leu Met Thr Gln Lys Thr Gly Ile Val Ser Gly PheGly Arg Thr His 355 360 365 GAG AAG GGC CGG CAG TCC ACC AGG CTC AAG ATGCTG GAG GTG CCC TA 1152 Glu Lys Gly Arg Gln Ser Thr Arg Leu Lys Met LeuGlu Val Pro Tyr 370 375 380 GTG GAC CGC AAC AGC TGC AAG CTG TCC AGC AGCTTC ATC ATC ACC CA 1200 Val Asp Arg Asn Ser Cys Lys Leu Ser Ser Ser PheIle Ile Thr Gln 385 390 395 400 AAC ATG TTC TGT GCC GGC TAC GAC ACC AAGCAG GAG GAT GCC TGC CA 1248 Asn Met Phe Cys Ala Gly Tyr Asp Thr Lys GlnGlu Asp Ala Cys Gln 405 410 415 GGG GAC AGC GGG GGC CCG CAC GTC ACC CGCTTC AAG GAC ACC TAC TT 1296 Gly Asp Ser Gly Gly Pro His Val Thr Arg PheLys Asp Thr Tyr Phe 420 425 430 GTG ACA GGC ATC GTC AGC TGG GGA GAG AGCTGT GCC CGT AAG GGG AA 1344 Val Thr Gly Ile Val Ser Trp Gly Glu Ser CysAla Arg Lys Gly Lys 435 440 445 TAC GGG ATC TAC ACC AAG GTC ACC GCC TTCCTC AAG TGG ATC GAC AG 1392 Tyr Gly Ile Tyr Thr Lys Val Thr Ala Phe LeuLys Trp Ile Asp Arg 450 455 460 TCC ATG AAA ACC AGG GGC TTG CCC AAG GCCAAG AGC CAT GCC CCG GA 1440 Ser Met Lys Thr Arg Gly Leu Pro Lys Ala LysSer His Ala Pro Glu 465 470 475 480 GTC ATA ACG TCC TCT CCA TTA AAG TGA1467 Val Ile Thr Ser Ser Pro Leu Lys 485 488 amino acids amino acidsingle linear protein 44 Met Gly Arg Pro Leu His Leu Val Leu Leu Ser AlaSer Leu Ala Gly 1 5 10 15 Leu Leu Leu Leu Gly Glu Ser Leu Phe Ile ArgArg Glu Gln Ala Asn 20 25 30 Asn Ile Leu Ala Arg Val Thr Arg Ala Asn SerPhe Leu Glu Glu Met 35 40 45 Lys Lys Gly His Leu Glu Arg Glu Cys Met GluGlu Thr Cys Ser Tyr 50 55 60 Glu Glu Ala Arg Glu Val Phe Glu Asp Ser AspLys Thr Asn Glu Phe 65 70 75 80 Trp Asn Lys Tyr Lys Asp Gly Asp Gln CysGlu Thr Ser Pro Cys Gln 85 90 95 Asn Gln Gly Lys Cys Lys Asp Gly Leu GlyGlu Tyr Thr Cys Thr Cys 100 105 110 Leu Glu Gly Phe Glu Gly Lys Asn CysGlu Leu Phe Thr Arg Lys Leu 115 120 125 Cys Ser Leu Asp Asn Gly Asp CysAsp Gln Phe Cys His Glu Glu Gln 130 135 140 Asn Ser Val Val Cys Ser CysAla Arg Gly Tyr Thr Leu Ala Asp Asn 145 150 155 160 Gly Lys Ala Cys IlePro Thr Gly Pro Tyr Pro Cys Gly Lys Gln Thr 165 170 175 Leu Glu Arg ArgLys Arg Ser Val Ala Gln Ala Thr Ser Ser Ser Gly 180 185 190 Glu Ala ProAsp Ser Ile Thr Trp Lys Pro Tyr Asp Ala Ala Asp Leu 195 200 205 Asp ProThr Glu Asn Pro Phe Asp Leu Leu Asp Phe Asn Gln Thr Gln 210 215 220 ProGlu Arg Gly Asp Asn Asn Leu Thr Arg Ile Val Gly Gly Gln Glu 225 230 235240 Cys Lys Asp Gly Glu Cys Pro Trp Gln Ala Leu Leu Ile Asn Glu Glu 245250 255 Asn Glu Gly Phe Cys Gly Gly Thr Ile Leu Ser Glu Phe Tyr Ile Leu260 265 270 Thr Ala Ala His Cys Leu Tyr Gln Ala Lys Arg Phe Lys Val ArgVal 275 280 285 Gly Asp Arg Asn Thr Glu Gln Glu Glu Gly Gly Glu Ala ValHis Glu 290 295 300 Val Glu Val Val Ile Lys His Asn Arg Phe Thr Lys GluThr Tyr Asp 305 310 315 320 Phe Asp Ile Ala Val Leu Arg Leu Lys Thr ProIle Thr Phe Arg Met 325 330 335 Asn Val Ala Pro Ala Cys Leu Pro Glu ArgAsp Trp Ala Glu Ser Thr 340 345 350 Leu Met Thr Gln Lys Thr Gly Ile ValSer Gly Phe Gly Arg Thr His 355 360 365 Glu Lys Gly Arg Gln Ser Thr ArgLeu Lys Met Leu Glu Val Pro Tyr 370 375 380 Val Asp Arg Asn Ser Cys LysLeu Ser Ser Ser Phe Ile Ile Thr Gln 385 390 395 400 Asn Met Phe Cys AlaGly Tyr Asp Thr Lys Gln Glu Asp Ala Cys Gln 405 410 415 Gly Asp Ser GlyGly Pro His Val Thr Arg Phe Lys Asp Thr Tyr Phe 420 425 430 Val Thr GlyIle Val Ser Trp Gly Glu Ser Cys Ala Arg Lys Gly Lys 435 440 445 Tyr GlyIle Tyr Thr Lys Val Thr Ala Phe Leu Lys Trp Ile Asp Arg 450 455 460 SerMet Lys Thr Arg Gly Leu Pro Lys Ala Lys Ser His Ala Pro Glu 465 470 475480 Val Ile Thr Ser Ser Pro Leu Lys 485 8 amino acids amino acid singlelinear peptide Modified-site 2...2 Xaa = Arg, Asp, Phe, Thr, Leu or Ser45 Gly Xaa Xaa Xaa Xaa Xaa Arg Xaa 1 5 8 amino acids amino acid singlelinear peptide 46 Gly Asp Asn Asn Leu Thr Arg Ile 1 5 8 amino acidsamino acid single linear peptide 47 Gly Asp Gln Asn Leu Thr Arg Ile 1 58 amino acids amino acid single linear peptide 48 Gly Lys Asn Asn LeuThr Arg Ile 1 5 8 amino acids amino acid single linear peptide 49 GlyLys Gln Asn Leu Thr Arg Ile 1 5 8 amino acids amino acid single linearpeptide 50 Gly Phe Asn Asp Phe Thr Arg Val 1 5 8 amino acids amino acidsingle linear peptide 51 Gly Phe Gln Asp Phe Thr Arg Val 1 5 8 aminoacids amino acid single linear peptide 52 Gly Lys Asn Asp Phe Thr ArgVal 1 5 8 amino acids amino acid single linear peptide 53 Gly Lys GlnAsp Phe Thr Arg Val 1 5 8 amino acids amino acid single linear peptide54 Gly Phe Asn Asp Phe Thr Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 55 Gly Phe Gln Asp Phe Thr Arg Ile 1 5 8 amino acidsamino acid single linear peptide 56 Gly Lys Asn Asp Phe Thr Arg Ile 1 58 amino acids amino acid single linear peptide 57 Gly Lys Gln Asp PheThr Arg Ile 1 5 8 amino acids amino acid single linear peptideModified-site 8...8 Xaa = Ile or Val 58 Gly Thr Lys Ile Lys Pro Arg Xaa1 5 8 amino acids amino acid single linear peptide Modified-site 8...8Xaa = Ile or Val 59 Gly Thr Gln Ile Lys Pro Arg Xaa 1 5 8 amino acidsamino acid single linear peptide Modified-site 8...8 Xaa = Ile or Val 60Gly Lys Lys Ile Lys Pro Arg Xaa 1 5 8 amino acids amino acid singlelinear peptide Modified-site 8...8 Xaa = Ile or Val 61 Gly Lys Gln IleLys Pro Arg Xaa 1 5 8 amino acids amino acid single linear peptide 62Gly Thr Lys Thr Ser Thr Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 63 Gly Thr Gln Thr Ser Thr Arg Ile 1 5 8 amino acidsamino acid single linear peptide 64 Gly Lys Lys Thr Ser Thr Arg Ile 1 58 amino acids amino acid single linear peptide 65 Gly Lys Gln Thr SerThr Arg Ile 1 5 8 amino acids amino acid single linear peptideModified-site 8...8 Xaa = Ile or Val 66 Gly Leu Ser Ser Met Thr Arg Xaa1 5 8 amino acids amino acid single linear peptide Modified-site 8...8Xaa = Ile or Val 67 Gly Leu Gln Ser Met Thr Arg Xaa 1 5 8 amino acidsamino acid single linear peptide Modified-site 8...8 Xaa = Ile or Val 68Gly Lys Ser Ser Met Thr Arg Xaa 1 5 8 amino acids amino acid singlelinear peptide Modified-site 8...8 Xaa = Ile or Val 69 Gly Lys Gln SerMet Thr Arg Xaa 1 5 8 amino acids amino acid single linear peptide 70Gly Ser Lys Pro Gln Gly Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 71 Gly Ser Gln Pro Gln Gly Arg Ile 1 5 8 amino acidsamino acid single linear peptide 72 Gly Lys Lys Pro Gln Gly Arg Ile 1 58 amino acids amino acid single linear peptide 73 Gly Lys Gln Pro GlnGly Arg Ile 1 5 8 amino acids amino acid single linear peptide 74 GlyLys Gln Ile Glu Gly Arg Ile 1 5 8 amino acids amino acid single linearpeptide 75 Gly Lys Gln Met Lys Thr Arg Ile 1 5 8 amino acids amino acidsingle linear peptide 76 Gly Leu Glu Arg Arg Lys Arg Ile 1 5 8 aminoacids amino acid single linear peptide 77 Gly Leu Gln Arg Arg Lys ArgIle 1 5 8 amino acids amino acid single linear peptide 78 Gly Lys GluArg Arg Lys Arg Ile 1 5 8 amino acids amino acid single linear peptide79 Gly Lys Gln Arg Arg Lys Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 80 Gly Leu Ala Arg Val Thr Arg Ile 1 5 8 amino acidsamino acid single linear peptide 81 Gly Leu Gln Arg Val Thr Arg Ile 1 58 amino acids amino acid single linear peptide 82 Gly Lys Ala Arg ValThr Arg Ile 1 5 8 amino acids amino acid single linear peptide 83 GlyLys Gln Arg Val Thr Arg Ile 1 5 8 amino acids amino acid single linearpeptide 84 Gly Leu Gln Arg Val Arg Arg Ile 1 5 8 amino acids amino acidsingle linear peptide 85 Gly Lys Gln Arg Val Arg Arg Ile 1 5 8 aminoacids amino acid single linear peptide 86 Gly Leu His Arg Arg Arg ArgIle 1 5 8 amino acids amino acid single linear peptide 87 Gly Leu GlnArg Arg Arg Arg Ile 1 5 8 amino acids amino acid single linear peptide88 Gly Lys His Arg Arg Arg Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 89 Gly Lys Gln Arg Arg Arg Arg Ile 1 5 8 amino acidsamino acid single linear peptide 90 Gly Leu Asn Arg Pro Lys Arg Ile 1 58 amino acids amino acid single linear peptide 91 Gly Leu Gln Arg ProLys Arg Ile 1 5 8 amino acids amino acid single linear peptide 92 GlyLys Asn Arg Pro Lys Arg Ile 1 5 8 amino acids amino acid single linearpeptide 93 Gly Lys Gln Arg Pro Lys Arg Ile 1 5 8 amino acids amino acidsingle linear peptide 94 Gly Leu Arg Ile Arg Lys Arg Ile 1 5 8 aminoacids amino acid single linear peptide 95 Gly Leu Gln Ile Arg Lys ArgIle 1 5 8 amino acids amino acid single linear peptide 96 Gly Lys ArgIle Arg Lys Arg Ile 1 5 8 amino acids amino acid single linear peptide97 Gly Lys Gln Ile Arg Lys Arg Ile 1 5 8 amino acids amino acid singlelinear peptide 98 Gly Lys Gln Arg Ser Lys Arg Ile 1 5 8 amino acidsamino acid single linear peptide 99 Gly Lys Gln Arg Val Thr Arg Ile 1 58 amino acids amino acid single linear peptide 100 Gly Lys Gln Arg LeuLys Arg Ile 1 5 5 amino acids amino acid single linear peptide 101 ArgVal Thr Arg Ile 1 5 10 amino acids amino acid single linear peptide 102Thr Lys Glu Arg Arg Lys Arg Ser Val Ala 1 5 10 8 amino acids amino acidsingle linear peptide 103 Asn Leu Thr Arg Ile Val Gly Gly 1 5 8 aminoacids amino acid single linear peptide 104 Asp Phe Thr Arg Val Val GlyGly 1 5 8 amino acids amino acid single linear peptide 105 Thr Leu GluArg Thr Val Gly Gly 1 5 8 amino acids amino acid single linear peptide106 Ile Lys Pro Arg Ile Val Gly Gly 1 5 8 amino acids amino acid singlelinear peptide 107 Ser Met Thr Arg Ile Val Gly Gly 1 5 8 amino acidsamino acid single linear peptide 108 Met Lys Thr Arg Ile Val Gly Gly 1 58 amino acids amino acid single linear peptide 109 Ile Glu Gly Arg IleVal Gly Gly 1 5 8 amino acids amino acid single linear peptide 110 ArgArg Lys Arg Ile Val Gly Gly 1 5 8 amino acids amino acid single linearpeptide 111 Arg Val Arg Arg Ile Val Gly Gly 1 5 8 amino acids amino acidsingle linear peptide 112 Arg Arg Arg Arg Ile Val Gly Gly 1 5 8 aminoacids amino acid single linear peptide 113 Arg Pro Lys Arg Ile Val GlyGly 1 5 8 amino acids amino acid single linear peptide 114 Ile Arg LysArg Ile Val Gly Gly 1 5 8 amino acids amino acid single linear peptide115 Arg Ser Lys Arg Ile Val Gly Gly 1 5 8 amino acids amino acid singlelinear peptide 116 Arg Val Thr Arg Ile Val Gly Gly 1 5 8 amino acidsamino acid single linear peptide 117 Arg Leu Lys Arg Ile Val Gly Gly 1 58 amino acids amino acid single linear peptide 118 Pro Gln Gly Arg IleVal Gly Gly 1 5 8 amino acids amino acid single linear peptide 119 ThrSer Thr Arg Ile Val Gly Gly 1 5 4 amino acids amino acid single linearpeptide 120 Met Lys Thr Arg 1 8 amino acids amino acid single linearpeptide 121 Xaa Xaa Xaa Arg Xaa Val Gly Gly 1 5 5 amino acids amino acidsingle linear peptide 122 Met Lys Thr Lys Gly 1 5 10 amino acids aminoacid single linear peptide 123 Gly Glu Ser Leu Phe Ile Arg Arg Glu Gln 15 10 14 amino acids amino acid single linear peptide 124 Ile Leu Ala ArgVal Thr Arg Ala Asn Ser Phe Leu Glu Glu 1 5 10 7 amino acids amino acidsingle linear peptide 125 Ser Val Ala Gln Ala Thr Ser 1 5 7 amino acidsamino acid single linear peptide 126 Leu Phe Ile Arg Arg Glu Gln 1 5 7amino acids amino acid single linear peptide 127 Ala Asn Ser Phe Leu GluGlu 1 5 10 amino acids amino acid single linear peptide 128 Val Thr ArgAla Asn Ser Phe Leu Glu Glu 1 5 10 5 amino acids amino acid singlelinear peptide 129 Arg Val Thr Arg Ala 1 5 5 amino acids amino acidsingle linear peptide 130 Arg Arg Lys Arg Ser 1 5 5 amino acids aminoacid single linear peptide Modified-site 3...3 Xaa = Lys or Arg 131 ArgXaa Xaa Arg Xaa 1 5 5 amino acids amino acid single linear peptide 132Asp Phe Thr Arg Val 1 5 5 amino acids amino acid single linear peptide133 Ile Lys Pro Arg Ile 1 5 5 amino acids amino acid single linearpeptide 134 Ser Met Thr Arg Ile 1 5 5 amino acids amino acid singlelinear peptide 135 Met Lys Thr Arg Ile 1 5 5 amino acids amino acidsingle linear peptide 136 Ile Glu Gly Arg Ile 1 5 5 amino acids aminoacid single linear peptide 137 Arg Arg Lys Arg Ile 1 5 5 amino acidsamino acid single linear peptide 138 Arg Val Arg Arg Ile 1 5 5 aminoacids amino acid single linear peptide 139 Arg Arg Arg Arg Ile 1 5 5amino acids amino acid single linear peptide 140 Arg Pro Lys Arg Ile 1 55 amino acids amino acid single linear peptide 141 Ile Arg Lys Arg Ile 15 5 amino acids amino acid single linear peptide 142 Arg Ser Lys Arg Ile1 5 5 amino acids amino acid single linear peptide 143 Arg Leu Lys ArgIle 1 5 5 amino acids amino acid single linear peptide 144 Pro Gln GlyArg Ile 1 5 5 amino acids amino acid single linear peptide 145 Thr SerThr Arg Ile 1 5

1. Factor XΔ analogue, characterized in that it has a deletion of aminoacids Arg180 to Arg234 of the factor XΔ amino acid sequence and amodification in the region of the amino acid sequence between Gly173 andArg179.
 2. Factor XΔ analogue according to claim 1, characterized inthat said modification represents a processing site of a protease notnaturally cleaving at this position of the factor X sequence.
 3. FactorXΔ analogue according to claim 1 or 2, characterized in that saidmodification is, at least, an amino acid exchange in the region of theamino acid sequence between Gly173 and Arg179, based on the amino acidnumbering as shown in FIG.
 1. 4. Factor XΔ analogue according to any oneof claims 1 to 3, characterized in that it contains a factor X sequencehaving Gly173-R6-R5-R4-R3-R2-Arg179/R1(235), wherein a) R1 is an aminoacid selected from the group of Val, Ser, Thr, Ile or Ala, b) R2 is anamino acid selected from the group of Glu, Thr, Pro, Gly, Lys or Arg, c)R3 is an amino acid selected from the group of Leu, Phe, Lys, Met, Gln,Glu, Ser, Val, Arg or Pro, d) R4 is an amino acid selected from thegroup of Thr, Asp, Asn, Ile, Ser, Met, Pro, Arg or Lys, e) R5 is anamino acid selected from the group of Asn, Lys, Ser, Glu, Gln, Ala, Hisor Arg, and f) R6 is an amino acid selected from the group of Asp, Phe,Thr, Arg, Leu or Ser.
 5. Factor XΔ analogue according to any oneof)claims 1 to 4, characterized in that said modification represents aprocessing site for a protease selected from the group of endoproteases,such as kexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7,serine proteases, such as factor IIa, factor VIIa, factor IXa, factorXIIa, factor XIa, factor Xa, or kallikrein, or a derivative of theseproteases.
 6. Factor XΔ analogue according to any one of claims 1 to 5,characterized in that it is present as a single chain molecule inenzymatically inactive form.
 7. Factor XΔ analogue according to any oneof claims 1 to 6, characterized in that said modification allowsactivation of the inactive, single chain factor XΔ analogue polypeptideto the double chain, active factor Xa analogue form.
 8. Factor XΔanalogue according to any one of claims 1 to 7, characterized in that ithas a further modification in the region of the C-terminal factor Xamino acid sequence.
 9. Factor XΔ analogue according to claim 8,characterized in that it has a modification in the C-terminal region ofthe β-peptide cleavage site.
 10. Factor XΔ analogue according to claim9, characterized in that said modification is a mutation, deletion orinsertion in the region of the factor X amino acid sequence betweenamino acid positions Arg469 and Ser476.
 11. Factor XΔ analogue accordingto any one of claims 8 to 10, characterized in that said modificationprevents the β-peptide from cleaving off.
 12. Factor XΔ analogueaccording to claim 8, characterized in that it has a deletion of thefactor X β-peptide.
 13. Factor XΔ analogue according to claim 12,characterized in that it has a translation stop signal in the C-terminalregion of the factor X sequence.
 14. Factor XΔ analogue according toclaim 13, characterized in that it has a translation stop signal at theposition of amino acid Lys470 of the factor X sequence.
 15. Factor XΔanalogue according to any one of claims 1 to 14, characterized in thatsaid modification in the region of the amino acid sequence betweenGly173 and Arg179 allows activation of the inactive factor X analogue toactive factor XΔ analogue in vitro.
 16. Factor XΔ analogue according toclaim 15, characterized in that said modification allows activation by aprotease selected from the group of endoproteases, such as kexin/Kex2,furin/PACE, PC1/PC3, PC2, PC4, PACE 4, LPC/PC7, serine proteases, suchas factor IIa, factor VIIa, factor IXa, factor XIIa, factor XIa, factorXa, or kallikrein, or a derivative of these proteases.
 17. Factor XΔanalogue according to any one of claims 1 to 16, characterized in thatit has an intact β-peptide and is provided as factor XΔα.
 18. Factor XΔanalogue according to any one of claims 1 to 16, characterized in thatit has a deletion of the β-peptide.
 19. Recombinant DNA coding for afactor XΔ analogue according to any one of claims 1 to 18, contained ina vector for recombinant expression of the encoded protein. 20.Transformed cells containing a recombinant DNA according to claim 19.21. Preparation containing purified factor XΔ analogue, which has adeletion of amino acids Arg180 to Arg234 of the factor X amino acidsequence and a modification in the region of the amino acid sequencebetween Gly173 and Arg179.
 22. Preparation according to claim 21,characterized in that it contains a single chain factor XΔ analogue inenzymatically inactive form having a purity of at least 80%, preferably90%, particularly preferably 95%, and that it does not contain anyinactive, proteolytic intermediates of factor X/Xa analogue. 23.Preparation according to any one of claims 21 or 22, characterized inthat it contains factor XΔ analogue as factor XΔα.
 24. Preparationaccording to any one of claims 21 or 22, characterized in that itcontains factor XΔ analogue as FXΔβ.
 25. Preparation according to anyone of claims 21 to 24, characterized in that it contains factor XΔanalogue as a single chain molecule in isolated form.
 26. Preparationaccording to any one of claims 21 to 25, characterized in that itcontains a factor XΔ analogue having high stability and structuralintegrity of the molecule.
 27. Preparation according to any one ofclaims 21 to 26, characterized in that it contains a factor XΔ analoguehaving a modification which allows activation of factor XΔ analogue tofactor Xa analogue in vitro.
 28. Preparation according to any one ofclaims 21 to 27, characterized in that it is formulated as apharmaceutical preparation.
 29. Preparation according to claim 28,characterized in that it is present in an appropriate device, preferablyan application device, in combination with a protease selected from thegroup of endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2,PC4, PACE 4, LPC/PC7, serine proteases, such as factor IIa, factor VIIa,factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or aderivative of these proteases.
 30. Preparation according to claim 29,characterized in that the components are provided spatially separated.31. Preparation containing a purified factor Xa analogue having highstability and structural integrity, which is particularly free ofinactive factor XΔ/Xa analogue intermediates and autoproteolyticdegradation products, obtainable by activating a factor XΔ analogueaccording to any one of claims 1 to
 18. 32. Preparation according toclaim 31, characterized in that it contains an active factor Xa analogueas a double chain molecule in isolated form.
 33. Preparation accordingto any one of claims 31 or 32, characterized in that it contains factorXa analogue having a purity of at least 80%, preferably 90%,particularly preferably 95%, and does not contain any inactive,proteolytic intermediates of factor X/Xa analogue.
 34. Preparationaccording to any one of claims 31 to 33, characterized in that itcontains a physiologically acceptable carrier and is provided in stablystorable form.
 35. Preparation according to any one of claims 21 to 34,characterized in that it optionally contains a blood factor or anactivated form of a blood factor as a further component.
 36. Preparationaccording to claim 35, characterized in that it contains at least onecomponent having factor VIII bypass activity as a further component. 37.Preparation according to any one of claims 21 or 36, characterized inthat it is formulated as a pharmaceutical composition.
 38. Use of apreparation according to any one of claims 21 to 37 for preparing amedicament.
 39. Use of a preparation according to any one of claims 21to 37 for preparing a medicament for the treatment of patients sufferingfrom blood coagulation disorders, such as patients suffering fromhemophilia or hemophiliacs who have developed inhibitor antibodies. 40.Process for preparing a preparation containing purified recombinantfactor XΔ analogue, characterized in that a factor XΔ analogue obtainedby recombinant preparation is isolated as a single chain molecule andpurified by means of a chromatographic process.
 41. Process according toclaim 40, characterized in that it comprises the following steps:providing a nucleic acid encoding a factor XΔ analogue according to anyone of claims 1 to 18 transfection of an appropriate cell expression ofthe factor XΔ analogue isolation of the single chain factor XΔ analogue,and purification of the polypeptide.
 42. Process for preparing apreparation containing active factor Xa analogue, characterized in thata preparation prepared according to any one of claims 40 or 41 issubjected to an activation step.
 43. Process according to claim 42,characterized in that the preparation containing single chain factor XΔanalogue is brought into contact with a protease selected from the groupof endoproteases, such as kexin/Kex2, furin/PACE, PC1/PC3, PC2, PC4,PACE 4, LPC/PC7, serine proteases, such as factor IIa, factor VIIa,factor IXa, factor XIIa, factor XIa, factor Xa, or kallikrein, or aderivative of these proteases, under conditions allowing cleavage to thedouble chain factor Xa analogue form.
 44. Process according to claim 43,characterized in that the protease is immobilized.
 45. Process accordingto any one of claims 41 to 44, characterized in that a purified factorXa analogue having high stability and structural integrity is obtained,which is particularly free of inactive factor XΔ/Xa analogueintermediates.